Compositions and methods for inhibiting hepcidin antimicrobial peptide (HAMP) or HAMP-related gene expression

ABSTRACT

The invention relates to lipid formulated double-stranded ribonucleic acid (dsRNA) targeting a hepcidin antimicrobial peptide (HAMP) and/or HAMP-related gene, and methods of using the dsRNA to inhibit expression of HAMP and/or HAMP-related genes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/118,489, filed Nov. 18, 2013, now allowed, which is a national stageentry of PCT application Serial No. PCT/US2012/043603, filed Jun. 21,2012, which claims the benefit of U.S. Provisional Application Ser. No.61/499,516, filed Jun. 21, 2011, and claims the benefit of U.S.Provisional Application Ser. No. 61/569,054, filed Dec. 9, 2011; each ofwhich are incorporated herein by reference, in their entirety, for allpurposes.

REFERENCE TO SEQUENCE LISTING

This application includes a Sequence Listing submitted electronically asa text file named 31128_US_CRF_sequencelisting.txt, created on Oct. 23,2015, with a size of 520,192 bytes. The sequence listing is incorporatedby reference.

FIELD

The disclosure relates to double-stranded ribonucleic acid (dsRNA)targeting HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1,and methods of using dsRNA to inhibit expression of HAMP, HFE2, HFE,TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1.

BACKGROUND

The discovery of the hepcidin peptide and characterization of its gene,HAMP, has led to the revision of previous models for the regulation ofiron homeostasis and the realization that the liver plays a key role indetermining iron absorption from the gut and iron release from recyclingand storage sites. In summary, the hepcidin model proposes that the rateof iron efflux into the plasma depends primarily on the plasma level ofhepcidin; when iron levels are high the synthesis of hepcidin increasesand the release of iron from enterocytes and macrophages is diminished.Conversely when iron stores drop, the synthesis of hepcidin isdown-regulated and these cells release more iron. Hepcidin directlybinds to ferroportin and decreases its functional activity by causing itto be internalized from the cell surface and degraded.

Hepcidin provides a unifying hypothesis to explain the behavior of ironin two diverse but common clinical conditions, the anemia of chronicdisease and both HFE and non-HFE haemochromatosis. The pathophysiologyof hepcidin has been sufficiently elucidated to offer promise oftherapeutic intervention in both of these situations. Administeringeither hepcidin or an agonist could treat haemochromatosis, where thesecretion of hepcidin is abnormally low.

The anemia of inflammation, commonly observed in patients with chronicinfections, malignancy, trauma, and inflammatory disorders, is awell-known clinical entity. Until recently, little was understood aboutits pathogenesis. It now appears that the inflammatory cytokine IL-6induces production of hepcidin, an iron-regulatory hormone that may beresponsible for most or all of the features of this disorder. (Andrews NC. J Clin Invest. 2004 May 1; 113(9): 1251-1253). As such, downregulation of hepcidin in anemic patients will lead to a reduction ininflammation associated with such anemia.

Double-stranded RNA molecules (dsRNA) have been shown to block geneexpression in a highly conserved regulatory mechanism known as RNAinterference (RNAi). WO 99/32619 (Fire et al.) discloses the use of adsRNA of at least 25 nucleotides in length to inhibit the expression ofgenes in C. elegans. dsRNA has also been shown to degrade target RNA inother organisms, including plants (see, e.g., WO 99/53050, Waterhouse etal.; and WO 99/61631, Heifetz et al.), Drosophila (see, e.g., Yang, D.,et al., Curr. Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895,Limmer; and DE 101 00 586.5, Kreutzer et al.). This natural mechanismhas now become the focus for the development of a new class ofpharmaceutical agents for treating disorders that are caused by theaberrant or unwanted regulation of a gene.

The following publications disclose dsRNA (siRNA) targeting the HAMPgene and are herein incorporated by reference for all purposes: WO2008/036933 (International application no. PCT/US2007/079212, filed Sep.21, 2007); US 2009-0209478 (U.S. patent application Ser. No. 11/859,288,filed Sep. 21, 2007); US 2010-0204307 (U.S. patent application Ser. No.12/757,497, filed Apr. 9, 2010); US 2011-0269823 (U.S. patentapplication Ser. No. 13/184,087, filed Jul. 15, 2011).

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the HAMP1 mRNA levels in mouse liver following variousdosages of siRNA and the serum iron concentration (μg/dL) followingvarious dosages of siRNA in mice.

FIG. 2 shows the HAMP mRNA levels in liver following siRNAadministration as well as the serum iron concentration (μg/dL) and theHAMP serum protein concentration (mg/mL) following siRNA administrationto non-human primates.

FIG. 3 shows the HAMP1 and TFR2 mRNA levels in mouse liver followingvarious dosages of siRNA and the percent (%) transferrin saturationfollowing various dosages of siRNA.

FIG. 4 shows the HAMP1 and TFR2 mRNA levels in mouse liver followingadministration of siRNA and the percent (%) transferrin saturation overa 30 day time course.

FIG. 5 shows the HAMP1 and TFR2 mRNA levels in rat liver followingadministration of siRNA. FIG. 5 also shows the serum iron and Hbconcentrations in rats at various time points.

FIG. 6 shows the level of HAMP mRNA reduction in the liver of eachanimal following siRNA administration, compared to PBS controls.

FIG. 7 shows the level of TFR2 mRNA reduction in the liver of eachanimal following siRNA administration, compared to PBS controls.

FIG. 8 shows that serum iron concentration was increased in each animalafter 1 mg/kg AD-52590 siRNA administration.

FIG. 9 shows that the HAMP serum protein concentration was decreased ineach animal following 1 mg/kg AD-52590 siRNA administration.

FIG. 10A shows combinatorial use of dsRNAs targeting differentHAMP-related mRNAs (HFE and TFR2) in vivo and relative mRNA levels forHFE (left bar), TFR2 (middle bar), and HAMP (right bar) for each group.

FIG. 10B shows UIBC (μg/dL) for each group.

FIG. 10C shows the percent transferring saturation for each group.

FIG. 10D shows serum iron concentration (μg/dL) for each group.

SUMMARY

Disclosed herein is a double-stranded ribonucleic acid (dsRNA) forinhibiting expression of hepcidin antimicrobial peptide (HAMP), whereinsaid dsRNA is selected from the dsRNAs listed in Table 2, 3, 4, or 5with a start position of 379, 380, 382, or 385. In some aspects, thedsRNA consists of a dsRNA listed in Table 2, 3, 4, or 5 with a startposition of 382.

Also described herein is a dsRNA for inhibiting expression of HAMP,wherein said dsRNA comprises a sense strand and an antisense strand, theantisense strand comprising a region of complementarity to a HAMP mRNAtranscript, wherein the antisense strand comprises at least 15contiguous nucleotides differing by no more than 3 nucleotides from oneof the antisense strand sequences listed in Table 2, 3, 4, or 5.

In some aspects, the region of complementarity is at least 17nucleotides in length. In some aspects, the region of complementarity isbetween 19 and 21 nucleotides in length. In some aspects, the region ofcomplementarity is 19 nucleotides in length. In some aspects, the regionof complementarity consists of one of the antisense strand sequences ofTable 2, 3, 4, or 5.

In some aspects, the sense strand comprises 15 or more contiguousnucleotides of one of the sense strand sequences of Table 2, 3, 4, or 5.In some aspects, the antisense strand comprises 15 or more contiguousnucleotides of one of the antisense strand sequences of Table 2, 3, 4,or 5. In some aspects, the sense strand comprises 15 or more contiguousnucleotides of one of the sense strand sequences of Table 2, 3, 4, or 5and the antisense strand comprises 15 or more contiguous nucleotides ofone of the antisense strand sequences of Table 2, 3, 4, or 5. In someaspects, the sense strand comprises 16, 17, 18, 19, 20, or morecontiguous nucleotides of one of the sense strand sequences of Table 2,3, 4, or 5 and the antisense strand comprises 16, 17, 18, 19, 20, ormore contiguous nucleotides of one of the antisense strand sequences ofTable 2, 3, 4, or 5. In some aspects, the sense strand comprises one ofthe sense strand sequences of Table 2, 3, 4, or 5. In some aspects, theantisense strand comprises one of the antisense strand sequences ofTable 2, 3, 4, or 5. In some aspects, the sense strand comprises one ofthe sense strand sequences of Table 2, 3, 4, or 5 and the antisensestrand comprises one of the antisense strand sequences of Table 2, 3, 4,or 5. In some aspects, the sense strand consists of one of the sensestrand sequences of Table 2, 3, 4, or 5 and the antisense strandconsists of one of the antisense strand sequences of Table 2, 3, 4, or5. In some aspects, the dsRNA mediates degradation of HAMP mRNA.

In some aspects, said dsRNA further comprises at least one modifiednucleotide. In some aspects, at least one of said modified nucleotidesis chosen from the group consisting of: a 2′-O-methyl modifiednucleotide, a nucleotide comprising a 5′-phosphorothioate group, and aterminal nucleotide linked to a cholesteryl derivative or dodecanoicacid bisdecylamide group. In some aspects, said modified nucleotide ischosen from the group consisting of: a 2′-fluoro modified nucleotide, a2′-fluoro modified nucleoside, a 2′-deoxy-2′-fluoro modified nucleotide,a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasicnucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide,morpholino nucleotide, a phosphoramidate, and a non-natural basecomprising nucleotide.

In some aspects, each strand is no more than 30 nucleotides in length.In some aspects, at least one strand comprises a 3′ overhang of at least1 nucleotide. In some aspects, at least one strand comprises a 3′overhang of at least 2 nucleotides. In some aspects, each strandcomprises a 3′ overhang of 2 nucleotides.

In some aspects, a dsRNA described above further comprises a ligand. Insome aspects, the ligand is conjugated to the 3′ end of the sense strandof the dsRNA. In some aspects, the dsRNA further comprises anN-Acetyl-Galactosamine (GalNac) conjugate.

In some aspects, a dsRNA described above is formulated in a nucleic acidlipid particle formulation. In some aspects, the nucleic acid lipidparticle formulation is selected from Table A. In some aspects, thenucleic acid lipid particle formulation comprises MC3.

Also described herein is a cell comprising a dsRNA described above.

Also described herein is a vector encoding at least one strand of adsRNA described above.

Also described herein is a cell comprising a vector described above.

Also described herein is a pharmaceutical composition for inhibitingexpression of a HAMP gene comprising a dsRNA described above. In someaspects, the composition further comprises a lipid formulation. In someaspects, the lipid formulation is a nucleic acid lipid particleformulation.

Also described herein is a dsRNA for inhibiting expression ofhemojuvelin (HFE2), wherein said dsRNA comprises a sense strand and anantisense strand, the antisense strand comprising a region ofcomplementarity to a HFE2 mRNA transcript, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by no more than 3nucleotides from one of the antisense strand sequences listed in Table10A.

In some aspects, the region of complementarity is at least 17nucleotides in length. In some aspects, the region of complementarity isbetween 19 and 21 nucleotides in length. In some aspects, the region ofcomplementarity is 19 nucleotides in length. In some aspects, the regionof complementarity consists of one of the antisense strand sequences ofTable 10A.

In some aspects, the sense strand comprises 15 or more contiguousnucleotides of one of the sense strand sequences of Table 10A. In someaspects, the antisense strand comprises 15 or more contiguousnucleotides of one of the antisense strand sequences of Table 10A. Insome aspects, the sense strand comprises 15 or more contiguousnucleotides of one of the sense strand sequences of Table 10A and theantisense strand comprises 15 or more contiguous nucleotides of one ofthe antisense strand sequences of Table 10A. In some aspects, the sensestrand comprises 16, 17, 18, 19, 20, or more contiguous nucleotides ofone of the sense strand sequences of Table 10A and the antisense strandcomprises 16, 17, 18, 19, 20, or more contiguous nucleotides of one ofthe antisense strand sequences of Table 10A. In some aspects, the sensestrand comprises one of the sense strand sequences of Table 10A. In someaspects, the antisense strand comprises one of the antisense strandsequences of Table 10A. In some aspects, the sense strand comprises oneof the sense strand sequences of Table 10A and the antisense strandcomprises one of the antisense strand sequences of Table 10A. In someaspects, the sense strand consists of one of the sense strand sequencesof Table 10A and the antisense strand consists of one of the antisensestrand sequences of Table 10A. In some aspects, the dsRNA mediatesdegradation of HFE2 mRNA.

In some aspects, said dsRNA further comprises at least one modifiednucleotide. In some aspects, at least one of said modified nucleotidesis chosen from the group consisting of: a 2′-O-methyl modifiednucleotide, a nucleotide comprising a 5′-phosphorothioate group, and aterminal nucleotide linked to a cholesteryl derivative or dodecanoicacid bisdecylamide group. In some aspects, said modified nucleotide ischosen from the group consisting of: a 2′-deoxy-2′-fluoro modifiednucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, anabasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modifiednucleotide, morpholino nucleotide, a phosphoramidate, and a non-naturalbase comprising nucleotide.

In some aspects, each strand is no more than 30 nucleotides in length.In some aspects, at least one strand comprises a 3′ overhang of at least1 nucleotide. In some aspects, at least one strand comprises a 3′overhang of at least 2 nucleotides. In some aspects, each strandcomprises a 3′ overhang of 2 nucleotides.

In some aspects, a dsRNA described above further comprises a ligand. Insome aspects, the ligand is conjugated to the 3′ end of the sense strandof the dsRNA. In some aspects, a dsRNA described above further comprisesa GalNac conjugate.

In some aspects, the dsRNA is formulated in a nucleic acid lipidparticle formulation. In some aspects, the nucleic acid lipid particleformulation is selected from Table A. In some aspects, the nucleic acidlipid particle formulation comprises MC3.

Also described herein is a cell comprising a dsRNA described above.

Also described herein is a vector encoding at least one strand of adsRNA described above.

Also described herein is a cell comprising a vector described above.

Also described herein is a pharmaceutical composition for inhibitingexpression of a HFE2 gene comprising a dsRNA described above. In someaspects, the composition further comprises a lipid formulation. In someaspects, the lipid formulation is a nucleic acid lipid particleformulation.

Also described herein is a dsRNA for inhibiting expression oftransferrin receptor 2 (TFR2), wherein said dsRNA comprises a sensestrand and an antisense strand, the antisense strand comprising a regionof complementarity to a TFR2 mRNA transcript, wherein the antisensestrand comprises at least 15 contiguous nucleotides differing by no morethan 3 nucleotides from one of the antisense strand sequences listed inTable 10B or 13.

In some aspects, the region of complementarity is at least 17nucleotides in length. In some aspects, the region of complementarity isbetween 19 and 21 nucleotides in length. In some aspects, the region ofcomplementarity is 19 nucleotides in length. In some aspects, the regionof complementarity consists of one of the antisense strand sequences ofTable 10B or 13.

In some aspects, the sense strand comprises 15 or more contiguousnucleotides of one of the sense strand sequences of Table 10B or 13. Insome aspects, the antisense strand comprises 15 or more contiguousnucleotides of one of the antisense strand sequences of Table 10B or 13.In some aspects, the sense strand comprises 15 or more contiguousnucleotides of one of the sense strand sequences of Table 10B or 13 andthe antisense strand comprises 15 or more contiguous nucleotides of oneof the antisense strand sequences of Table 10B or 13. In some aspects,the sense strand comprises 16, 17, 18, 19, 20, or more contiguousnucleotides of one of the sense strand sequences of Table 10B or 13 andthe antisense strand comprises 16, 17, 18, 19, 20, or more contiguousnucleotides of one of the antisense strand sequences of Table 10B or 13.In some aspects, sense strand comprises one of the sense strandsequences of Table 10B or 13. In some aspects, the antisense strandcomprises one of the antisense strand sequences of Table 10B or 13. Insome aspects, the sense strand comprises one of the sense strandsequences of Table 10B or 13 and the antisense strand comprises one ofthe antisense strand sequences of Table 10B or 13. In some aspects, thesense strand consists of one of the sense strand sequences of Table 10Bor 13 and the antisense strand consists of one of the antisense strandsequences of Table 10B or 13. In some aspects, the dsRNA mediatesdegradation of TFR2 mRNA.

In some aspects, said dsRNA further comprises at least one modifiednucleotide. In some aspects, at least one of said modified nucleotidesis chosen from the group consisting of: a 2′-O-methyl modifiednucleotide, a nucleotide comprising a 5′-phosphorothioate group, and aterminal nucleotide linked to a cholesteryl derivative or dodecanoicacid bisdecylamide group. In some aspects, said modified nucleotide ischosen from the group consisting of: a 2′-deoxy-2′-fluoro modifiednucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, anabasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modifiednucleotide, morpholino nucleotide, a phosphoramidate, and a non-naturalbase comprising nucleotide.

In some aspects, each strand is no more than 30 nucleotides in length.In some aspects, at least one strand comprises a 3′ overhang of at least1 nucleotide. In some aspects, at least one strand comprises a 3′overhang of at least 2 nucleotides. In some aspects, each strandcomprises a 3 overhang of 2 nucleotides.

In some aspects, a dsRNA described above further comprises a ligand. Insome aspects, the ligand is conjugated to the 3′ end of the sense strandof the dsRNA. In some aspects, a dsRNA described above further comprisesa GalNac conjugate.

In some aspects, the dsRNA is formulated in a nucleic acid lipidparticle formulation. In some aspects, the nucleic acid lipid particleformulation is selected from Table A. In some aspects, the nucleic acidlipid particle formulation comprises MC3.

Also described herein is a cell comprising a dsRNA described above.

Also described herein is a vector encoding at least one strand of adsRNA described above.

Also described herein is a cell comprising a vector described above.

Also described herein is a pharmaceutical composition for inhibitingexpression of a TFR2 gene comprising a dsRNA described above. In someaspects, the composition further comprises a lipid formulation. In someaspects, the lipid formulation is a nucleic acid lipid particleformulation.

Also described herein is a composition comprising a first dsRNA forinhibiting expression of a HAMP gene and a second dsRNA for inhibitingexpression of an HFE2 gene, wherein the first dsRNA comprises a firstsense strand and an first antisense strand, the first antisense strandcomprising a region of complementarity to a HAMP mRNA transcript,wherein the first antisense strand comprises at least 15 contiguousnucleotides differing by no more than 3 nucleotides from one of theantisense strand sequences listed in Table 2, 3, 4, or 5; and whereinthe second dsRNA comprises a second sense strand and a second antisensestrand, the second antisense strand comprising a region ofcomplementarity to a HFE2 mRNA transcript, wherein the second antisensestrand comprises at least 15 contiguous nucleotides differing by no morethan 3 nucleotides from one of the antisense strand sequences listed inTable 10A.

Also described herein is a composition comprising a first dsRNA forinhibiting expression of a HAMP gene and a second dsRNA for inhibitingexpression of an TFR2 gene, wherein said first dsRNA comprises a firstsense strand and a first antisense strand, the first antisense strandcomprising a region of complementarity to a HAMP mRNA transcript,wherein the first antisense strand comprises at least 15 contiguousnucleotides differing by no more than 3 nucleotides from one of theantisense strand sequences listed in Table 2, 3, 4, or 5; and whereinsaid second dsRNA comprises a second sense strand and a second antisensestrand, the second antisense strand comprising a region ofcomplementarity to a TFR2 mRNA transcript, wherein the second antisensestrand comprises at least 15 contiguous nucleotides differing by no morethan 3 nucleotides from one of the antisense strand sequences listed inTable 10B or 13.

Also described herein is a composition comprising a first dsRNA forinhibiting expression of a TFR2 gene and a second dsRNA for inhibitingexpression of a HFE2 gene, wherein said first dsRNA comprises a firstsense strand and a first antisense strand, the first antisense strandcomprising a region of complementarity to a TFR2 mRNA transcript,wherein the first antisense strand comprises at least 15 contiguousnucleotides differing by no more than 3 nucleotides from one of theantisense strand sequences listed in Table 10B or 13; and wherein saidsecond dsRNA comprises a second sense strand and a second antisensestrand, the second antisense strand comprising a region ofcomplementarity to a HFE2 mRNA transcript, wherein the second antisensestrand comprises at least 15 contiguous nucleotides differing by no morethan 3 nucleotides from one of the antisense strand sequences listed inTable 10A.

Also described herein is a composition comprising a plurality of dsRNAsselected from the dsRNAs described above.

Also described herein is a method of inhibiting HAMP expression in acell, the method comprising: (a) introducing into the cell a dsRNAdescribed above; and (b) maintaining the cell produced in step (a) for atime sufficient to obtain degradation of the mRNA transcript of a HAMPgene, thereby inhibiting expression of the HAMP gene in the cell. Insome aspects, the HAMP expression is inhibited by at least 30%. In someaspects, the HAMP expression is inhibited by at least 80%.

Also described herein is a method of treating a disorder associated withHAMP expression comprising administering to a subject in need of suchtreatment a therapeutically effective amount of a dsRNA described above.

In some aspects, the subject has anemia. In some aspects, the subjecthas refractory anemia. In some aspects, the subject has anemia ofchronic disease (ACD). In some aspects, the subject has iron-restrictederythropoiesis. In some aspects, the subject is a human.

In some aspects, the dsRNA is administered at a concentration of 0.01mg/kg-5 mg/kg bodyweight of the subject.

In some aspects, the dsRNA is lipid formulated. In some aspects, thedsRNA is lipid formulated in a formulation selected from Table A. Insome aspects, the dsRNA is lipid formulated in a nucleic acid lipidparticle formulation. In some aspects, the dsRNA is lipid formulated ina nucleic acid lipid particle formulation and administeredintravenously. In some aspects, the dsRNA is conjugated to GalNac. Insome aspects, the dsRNA is conjugated to GalNac and administeredsubcutaneously. In some aspects, the dsRNA is administeredsubcutaneously.

Also described herein is a method for treating anemia in a subject inneed thereof comprising administering to the subject an effective amountof a HAMP dsRNA described above.

In some aspects, the dsRNA is lipid formulated. In some aspects, thedsRNA is lipid formulated in a nucleic acid lipid particle formulation.In some aspects, the dsRNA is lipid formulated in a nucleic acid lipidparticle formulation and administered intravenously. In some aspects,the dsRNA is administered intravenously. In some aspects, the dsRNA islipid formulated in a formulation selected from Table A. In someaspects, the dsRNA is conjugated to GalNac. In some aspects, the dsRNAis conjugated to GalNac and administered subcutaneously. In someaspects, the dsRNA is administered subcutaneously.

In some aspects, the subject is a primate or a rodent. In some aspects,the subject is a human.

In some aspects, the effective amount is a concentration of 0.01-5.0mg/kg bodyweight of the subject.

In some aspects, the subject has fatigue, shortness of breath, headache,dizziness, or pale skin. In some aspects, the subject has reduced ironlevels compared to a subject without anemia. In some aspects, thesubject has haemoglobin (Hb) levels <9 g/dL. In some aspects, thesubject has chronic kidney disease (CKD), cancer, chronic inflammatorydisease, rheumatoid arthritis (RA), or iron-resistant iron-deficientamemia (IRIDA). In some aspects, the subject has reduced renalerythropoietin (EPO) synthesis compared to a subject without CKD, adietary deficiency, blood loss, or elevated hepcidin levels compared toa subject without CKD. In some aspects, the subject has decreased renalexcretion of hepcidin compared to a subject without CKD or low gradeinflammation characterized by increased interleukin-6 (IL-6) levelscompared to a subject without CKD. In some aspects, the subject has areticulocyte Hb of <28 pg. In some aspects, the subject has >10%hypochromic red blood cells (RBCs). In some aspects, the method furthercomprises determining the complete blood count (CBC), serum ironconcentration, Transferrin (Tf) saturation, or ferritin levels of thesubject.

In some aspects, administering results in an increase in iron levels inthe subject. In some aspects, administering results in a 2-fold increasein iron levels in the subject. In some aspects, administering results inan increase in Tf saturation in the subject.

In some aspects, the method further comprises determining the iron levelin the subject. In some aspects, the method further comprisesadministering intravenous iron or ESAs to the subject.

Also described herein is a method of inhibiting HFE2 expression in acell, the method comprising: (a) introducing into the cell a dsRNAdescribed above; and (b) maintaining the cell produced in step (a) for atime sufficient to obtain degradation of the mRNA transcript of a HFE2gene, thereby inhibiting expression of the HFE2 gene in the cell. Insome aspects, the HFE2 expression is inhibited by at least 30%. In someaspects, the HFE2 expression is inhibited by at least 80%.

Also described herein is a method of treating a disorder associated withHFE2 expression comprising administering to a subject in need of suchtreatment a therapeutically effective amount of a dsRNA described above.

In some aspects, the subject has anemia. In some aspects, the subjecthas refractory anemia. In some aspects, the subject has anemia ofchronic disease (ACD). In some aspects, the subject has iron-restrictederythropoiesis. In some aspects, the subject is a human.

In some aspects, the dsRNA is administered at a concentration of 0.01mg/kg-5 mg/kg bodyweight of the subject.

In some aspects, the dsRNA is lipid formulated. In some aspects, thedsRNA is lipid formulated in a formulation selected from Table A. Insome aspects, the dsRNA is lipid formulated in a nucleic acid lipidparticle formulation. In some aspects, the dsRNA is lipid formulated ina nucleic acid lipid particle formulation and administeredintravenously. In some aspects, the dsRNA is conjugated to GalNac. Insome aspects, the dsRNA is conjugated to GalNac and administeredsubcutaneously. In some aspects, the dsRNA is administeredsubcutaneously.

Also described herein is a method for treating anemia in a subject inneed thereof comprising administering to the subject an effective amountof a HFE2 dsRNA described above.

In some aspects, the dsRNA is lipid formulated. In some aspects, thedsRNA is lipid formulated in a nucleic acid lipid particle formulation.In some aspects, the dsRNA is lipid formulated in a nucleic acid lipidparticle formulation and administered intravenously. In some aspects,the dsRNA is administered intravenously. In some aspects, the dsRNA islipid formulated in a formulation selected from Table A. In someaspects, the dsRNA is conjugated to GalNac. In some aspects, the dsRNAis conjugated to GalNac and administered subcutaneously. In someaspects, the dsRNA is administered subcutaneously.

In some aspects, the subject is a primate or a rodent. In some aspects,the subject is a human.

In some aspects, the effective amount is a concentration of 0.01-5.0mg/kg bodyweight of the subject.

In some aspects, the subject has fatigue, shortness of breath, headache,dizziness, or pale skin. In some aspects, the subject has reduced ironlevels compared to a subject without anemia. In some aspects, thesubject has haemoglobin (Hb) levels <9 g/dL. In some aspects, thesubject has chronic kidney disease (CKD), cancer, chronic inflammatorydisease, rheumatoid arthritis (RA), or iron-resistant iron-deficientamemia (IRIDA). In some aspects, the subject has reduced renalerythropoietin (EPO) synthesis compared to a subject without CKD, adietary deficiency, blood loss, or elevated hepcidin levels compared toa subject without CKD. In some aspects, the subject has decreased renalexcretion of hepcidin compared to a subject without CKD or low gradeinflammation characterized by increased interleukin-6 (IL-6) levelscompared to a subject without CKD. In some aspects, the subject has areticulocyte Hb of <28 pg. In some aspects, the subject has >10%hypochromic red blood cells (RBCs). In some aspects, the method furthercomprises determining the complete blood count (CBC), serum ironconcentration, Transferrin (Tf) saturation, or ferritin levels of thesubject.

In some aspects, administering results in an increase in iron levels inthe subject. In some aspects, administering results in a 2-fold increasein iron levels in the subject. In some aspects, administering results inan increase in Tf saturation in the subject.

In some aspects, the method further comprises determining the iron levelin the subject. In some aspects, the method further comprisesadministering intravenous iron or ESAs to the subject.

Also described herein is a method of inhibiting TFR2 expression in acell, the method comprising: (a) introducing into the cell a dsRNAdescribed above; and (b) maintaining the cell produced in step (a) for atime sufficient to obtain degradation of the mRNA transcript of a TFR2gene, thereby inhibiting expression of the TFR2 gene in the cell. Insome aspects, the TFR2 expression is inhibited by at least 30%. In someaspects, the TFR2 expression is inhibited by at least 80%.

Also described herein is a method of treating a disorder associated withTFR2 expression comprising administering to a subject in need of suchtreatment a therapeutically effective amount of a dsRNA described above.

In some aspects, the subject has anemia. In some aspects, the subjecthas refractory anemia. In some aspects, the subject has anemia ofchronic disease (ACD). In some aspects, the subject has iron-restrictederythropoiesis. In some aspects, the subject is a human.

In some aspects, the dsRNA is administered at a concentration of 0.01mg/kg-5 mg/kg bodyweight of the subject.

In some aspects, the dsRNA is lipid formulated. In some aspects, thedsRNA is lipid formulated in a formulation selected from Table A. Insome aspects, the dsRNA is lipid formulated in a nucleic acid lipidparticle formulation. In some aspects, the dsRNA is lipid formulated ina nucleic acid lipid particle formulation and administeredintravenously. In some aspects, the dsRNA is conjugated to GalNac. Insome aspects, the dsRNA is conjugated to GalNac and administeredsubcutaneously. In some aspects, the dsRNA is administeredsubcutaneously.

Also described herein is a method for treating anemia in a subject inneed thereof comprising administering to the subject an effective amountof a TFR2 dsRNA described above.

In some aspects, the dsRNA is lipid formulated. In some aspects, thedsRNA is lipid formulated in a nucleic acid lipid particle formulation.In some aspects, the dsRNA is lipid formulated in a nucleic acid lipidparticle formulation and administered intravenously. In some aspects,the dsRNA is administered intravenously. In some aspects, the dsRNA islipid formulated in a formulation selected from Table A. In someaspects, the dsRNA is conjugated to GalNac. In some aspects, the dsRNAis conjugated to GalNac and administered subcutaneously. In someaspects, the dsRNA is administered subcutaneously.

In some aspects, the subject is a primate or a rodent. In some aspects,the subject is a human.

In some aspects, the effective amount is a concentration of 0.01-5.0mg/kg bodyweight of the subject.

In some aspects, the subject has fatigue, shortness of breath, headache,dizziness, or pale skin. In some aspects, the subject has reduced ironlevels compared to a subject without anemia. In some aspects, thesubject has haemoglobin (Hb) levels <9 g/dL. In some aspects, thesubject has chronic kidney disease (CKD), cancer, chronic inflammatorydisease, rheumatoid arthritis (RA), or iron-resistant iron-deficientamemia (IRIDA). In some aspects, the subject has reduced renalerythropoietin (EPO) synthesis compared to a subject without CKD, adietary deficiency, blood loss, or elevated hepcidin levels compared toa subject without CKD. In some aspects, the subject has decreased renalexcretion of hepcidin compared to a subject without CKD or low gradeinflammation characterized by increased interleukin-6 (IL-6) levelscompared to a subject without CKD. In some aspects, the subject has areticulocyte Hb of <28 pg. In some aspects, the subject has >10%hypochromic red blood cells (RBCs). In some aspects, the method furthercomprises determining the complete blood count (CBC), serum ironconcentration, Transferrin (Tf) saturation, or ferritin levels of thesubject.

In some aspects, administering results in an increase in iron levels inthe subject. In some aspects, administering results in a 2-fold increasein iron levels in the subject. In some aspects, administering results inan increase in Tf saturation in the subject.

In some aspects, the method further comprises determining the iron levelin the subject. In some aspects, the method further comprisesadministering intravenous iron or ESAs to the subject.

Also described herein is a method of inhibiting HAMP, HFE2, and/or TFR2expression in a cell, the method comprising: (a) introducing into thecell a plurality of dsRNAs selected from the dsRNAs described above; and(b) maintaining the cell produced in step (a) for a time sufficient toobtain degradation of the mRNA transcript of a HAMP, HFE2, and/or TFR2gene, thereby inhibiting expression of the HAMP, HFE2, and/or TFR2 genein the cell.

In some aspects, the plurality of dsRNAs are introduced simultaneously.In some aspects, the plurality of dsRNAs are introduced concurrently. Insome aspects, the plurality of dsRNAs are introduced individually. Insome aspects, the plurality of dsRNAs are introduced together. In someaspects, the expression is inhibited by at least 30%. In some aspects,the expression is inhibited by at least 80%.

Also described herein is a method of treating a disorder associated withHAMP, HFE2, and/or TFR2 expression comprising administering to a subjectin need of such treatment a therapeutically effective amount of aplurality of dsRNAs selected from the dsRNAs described above.

In some aspects, the plurality of dsRNAs are administered to the subjectsimultaneously. In some aspects, the plurality of dsRNAs areadministered to the subject concurrently. In some aspects, the pluralityof dsRNAs are administered to the subject individually. In some aspects,the plurality of dsRNAs are administered to the subject together.

In some aspects, the subject has anemia. In some aspects, the subjecthas refractory anemia. In some aspects, the subject has anemia ofchronic disease (ACD). In some aspects, the subject has iron-restrictederythropoiesis. In some aspects, the subject is a human. In someaspects, the plurality is administered at a concentration of 0.01mg/kg-5 mg/kg bodyweight of the subject.

Also described herein is a method for treating anemia in a subject inneed thereof comprising administering to the subject an effective amountof a plurality of dsRNAs selected from the dsRNAs described above.

In some aspects, the plurality of dsRNAs are administered to the subjectsimultaneously. In some aspects, the plurality of dsRNAs areadministered to the subject concurrently. In some aspects, the pluralityof dsRNAs are administered to the subject individually. In some aspects,the plurality of dsRNAs are administered to the subject together.

In some aspects, the plurality is lipid formulated. In some aspects, theplurality is lipid formulated in a nucleic acid lipid particleformulation. In some aspects, the plurality is lipid formulated in anucleic acid lipid particle formulation and administered intravenously.In some aspects, the plurality is administered intravenously. In someaspects, the plurality is lipid formulated in a formulation selectedfrom Table A. In some aspects, the plurality is conjugated to GalNac. Insome aspects, the plurality is conjugated to GalNac and administeredsubcutaneously. In some aspects, the plurality is administeredsubcutaneously.

In some aspects, the subject is a primate or a rodent. In some aspects,the subject is a human.

In some aspects, the effective amount is a concentration of 0.01-5.0mg/kg bodyweight of the subject.

In some aspects, the subject has fatigue, shortness of breath, headache,dizziness, or pale skin. In some aspects, the subject has reduced ironlevels compared to a subject without anemia. In some aspects, thesubject has haemoglobin (Hb) levels <9 g/dL. In some aspects, thesubject has chronic kidney disease (CKD), cancer, chronic inflammatorydisease, rheumatoid arthritis (RA), or iron-resistant iron-deficientamemia (IRIDA). In some aspects, the subject has reduced renalerythropoietin (EPO) synthesis compared to a subject without CKD, adietary deficiency, blood loss, or elevated hepcidin levels compared toa subject without CKD. In some aspects, the subject has decreased renalexcretion of hepcidin compared to a subject without CKD or low gradeinflammation characterized by increased interleukin-6 (IL-6) levelscompared to a subject without CKD. In some aspects, the subject has areticulocyte Hb of <28 pg. In some aspects, the subject has >10%hypochromic red blood cells (RBCs). In some aspects, the method furthercomprises determining the complete blood count (CBC), serum ironconcentration, Transferrin (Tf) saturation, or ferritin levels of thesubject.

In some aspects, administering results in an increase in iron levels inthe subject. In some aspects, administering results in a 2-fold increasein iron levels in the subject. In some aspects, administering results inan increase in Tf saturation in the subject.

In some aspects, the method further comprises determining the iron levelin the subject. In some aspects, the method further comprisesadministering intravenous iron or ESAs to the subject.

DETAILED DESCRIPTION

The details of one or more embodiments are set forth in the descriptionbelow. Other features, objects, and advantages will be apparent from thedescription and the drawings, and from the claims.

Provided herein are dsRNAs and methods of using the dsRNAs forinhibiting the expression of HAMP in a cell or a mammal where the dsRNAtargets HAMP. Also provided are compositions and methods for treatingpathological conditions and diseases in a mammal caused by theexpression of HAMP. A HAMP dsRNA directs the sequence-specificdegradation of HAMP mRNA.

Also provided herein are dsRNAs and methods of using the dsRNAs forinhibiting the expression of HFE2 in a cell or a mammal where the dsRNAtargets HFE2. Also provided are compositions and methods for treatingpathological conditions and diseases in a mammal caused by theexpression of HFE2. A HFE2 dsRNA directs the sequence-specificdegradation of HFE2 mRNA.

Also provided herein are dsRNAs and methods of using the dsRNAs forinhibiting the expression of HFE in a cell or a mammal where the dsRNAtargets HFE. Also provided are compositions and methods for treatingpathological conditions and diseases in a mammal caused by theexpression of HFE. A HFE dsRNA directs the sequence-specific degradationof HFE mRNA.

Also provided herein are dsRNAs and methods of using the dsRNAs forinhibiting the expression of TFR2 in a cell or a mammal where the dsRNAtargets TFR2. Also provided are compositions and methods for treatingpathological conditions and diseases in a mammal caused by theexpression of TFR2. A TFR2 dsRNA directs the sequence-specificdegradation of TFR2 mRNA.

Also provided herein are dsRNAs and methods of using the dsRNAs forinhibiting the expression of BMPR1a in a cell or a mammal where thedsRNA targets BMPR1a. Also provided are compositions and methods fortreating pathological conditions and diseases in a mammal caused by theexpression of BMPR1a. A BMPR1a dsRNA directs the sequence-specificdegradation of BMPR1a mRNA.

Also provided herein are dsRNAs and methods of using the dsRNAs forinhibiting the expression of SMAD4 in a cell or a mammal where the dsRNAtargets SMAD4. Also provided are compositions and methods for treatingpathological conditions and diseases in a mammal caused by theexpression of SMAD4. A SMAD4 dsRNA directs the sequence-specificdegradation of SMAD4 mRNA.

Also provided herein are dsRNAs and methods of using the dsRNAs forinhibiting the expression of IL6R in a cell or a mammal where the dsRNAtargets IL6R. Also provided are compositions and methods for treatingpathological conditions and diseases in a mammal caused by theexpression of IL6R. An IL6R dsRNA directs the sequence-specificdegradation of IL6R mRNA.

Also provided herein are dsRNAs and methods of using the dsRNAs forinhibiting the expression of BMP6 in a cell or a mammal where the dsRNAtargets BMP6. Also provided are compositions and methods for treatingpathological conditions and diseases in a mammal caused by theexpression of BMP6. A BMP6 dsRNA directs the sequence-specificdegradation of BMP6 mRNA.

Also provided herein are dsRNAs and methods of using the dsRNAs forinhibiting the expression of NEO1 in a cell or a mammal where the dsRNAtargets NEO1. Also provided are compositions and methods for treatingpathological conditions and diseases in a mammal caused by theexpression of NEO1. A NEO1 dsRNA directs the sequence-specificdegradation of NEO1 mRNA.

DEFINITIONS

For convenience, the meaning of certain terms and phrases used in thespecification, examples, and appended claims, are provided below. Ifthere is an apparent discrepancy between the usage of a term in otherparts of this specification and its definition provided in this section,the definition in this section shall prevail.

“G,” “C,” “A” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, and uracil as a base, respectively.“T” and “dT” are used interchangeably herein and refer to adeoxyribonucleotide wherein the nucleobase is thymine, e.g.,deoxyribothymine. However, it will be understood that the term“ribonucleotide” or “nucleotide” or “deoxyribonucleotide” can also referto a modified nucleotide, as further detailed below, or a surrogatereplacement moiety. The skilled person is well aware that guanine,cytosine, adenine, and uracil may be replaced by other moieties withoutsubstantially altering the base pairing properties of an oligonucleotidecomprising a nucleotide bearing such replacement moiety. For example,without limitation, a nucleotide comprising inosine as its base may basepair with nucleotides containing adenine, cytosine, or uracil. Hence,nucleotides containing uracil, guanine, or adenine may be replaced inthe nucleotide sequences of the invention by a nucleotide containing,for example, inosine. Sequences comprising such replacement moieties areembodiments of the invention.

As used herein, “HAMP” refers to the hepcidin antimicrobial peptidegene, transcript, or protein (also known as LEAP). A human mRNA sequencefor HAMP is Genbank accession NM_021175.2, included below as SEQ IDNO:1. Other examples of mammalian HAMP sequences are shown in Table B.

As used herein, “HFE2” refers to hemojuvelin gene, transcript, orprotein. Examples of mammalian HFE2 sequences are shown in Table B.

As used herein, “TFR2” refers to transferrin receptor 2 gene,transcript, or protein. Examples of mammalian TFR2 sequences are shownin Table B.

As used herein, “HFE” refers to hemochromatosis gene, transcript, orprotein. Examples of mammalian HFE sequences are shown in Table B.

As used herein, “BMPR1a” refers to bone morphogenetic protein receptor,type 1A gene, transcript, or protein. Examples of mammalian BMPR1asequences are shown in Table B.

As used herein, “SMAD4” refers to SMAD family member 4 gene, transcript,or protein. Examples of mammalian SMAD4 sequences are shown in Table B.

As used herein, “IL6R” refers to interleukin 6 receptor gene,transcript, or protein. Examples of mammalian IL6R sequences are shownin Table B.

As used herein, “BMP6” refers to bone morphogenetic protein 6 gene,transcript, or protein. Examples of mammalian BMP6 sequences are shownin Table B.

As used herein, “NEO1” refers to neogenin homolog 1 gene, transcript, orprotein. Examples of mammalian NEO1 sequences are shown in Table B.

As used herein, “HAMP-related” refers to a HFE2, HFE, TFR2, BMPR1a,SMAD4, IL6R, BMP6, and/or NEO1 gene, transcript, or protein.

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene,including mRNA that is a product of RNA processing of a primarytranscription product.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.Such conditions can, for example, be stringent conditions, wherestringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Otherconditions, such as physiologically relevant conditions as may beencountered inside an organism, can apply. The skilled person will beable to determine the set of conditions most appropriate for a test ofcomplementarity of two sequences in accordance with the ultimateapplication of the hybridized nucleotides.

This includes base-pairing of the oligonucleotide or polynucleotidecomprising the first nucleotide sequence to the oligonucleotide orpolynucleotide comprising the second nucleotide sequence over the entirelength of the first and second nucleotide sequence. Such sequences canbe referred to as “fully complementary” with respect to each otherherein. However, where a first sequence is referred to as complementarywith respect to a second sequence herein, the two sequences can be fullycomplementary, or they may be “substantially complementary,” e.g., theymay form one or more, but generally not more than 4, 3 or 2 mismatchedbase pairs upon hybridization, while retaining the ability to hybridizeunder the conditions most relevant to their ultimate application.However, where two oligonucleotides are designed to form, uponhybridization, one or more single stranded overhangs, such overhangsshall not be regarded as mismatches with regard to the determination ofcomplementarity. For example, a dsRNA comprising one oligonucleotide 21nucleotides in length and another oligonucleotide 23 nucleotides inlength, wherein the longer oligonucleotide comprises a sequence of 21nucleotides that is fully complementary to the shorter oligonucleotide,may yet be referred to as “fully complementary” for the purposesdescribed herein.

“Complementary” sequences, as used herein, may also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in as far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs includes, but not limited to, G:UWobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantiallycomplementary” herein may be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenthe antisense strand of a dsRNA and a target sequence, as will beunderstood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary toat least part of” a messenger RNA (mRNA) refers to a polynucleotide thatis substantially complementary to a contiguous portion of the mRNA ofinterest (e.g., an mRNA encoding HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4,IL6R, BMP6, and/or NEO1) including a 5′ UTR, an open reading frame(ORF), or a 3′ UTR. For example, a polynucleotide is complementary to atleast a part of a HAMP mRNA if the sequence is substantiallycomplementary to a non-interrupted portion of an mRNA encoding HAMP.

The term “double-stranded RNA” or “dsRNA,” as used herein, refers to acomplex of ribonucleic acid molecules, having a duplex structurecomprising two anti-parallel and substantially complementary, as definedabove, nucleic acid strands. In general, the majority of nucleotides ofeach strand are ribonucleotides, but as described in detail herein, eachor both strands can also include at least one non-ribonucleotide, e.g.,a deoxyribonucleotide and/or a modified nucleotide. In addition, as usedin this specification, “dsRNA” may include chemical modifications toribonucleotides, including substantial modifications at multiplenucleotides and including all types of modifications disclosed herein orknown in the art. Any such modifications, as used in an siRNA typemolecule, are encompassed by “dsRNA” for the purposes of thisspecification and claims.

The two strands forming the duplex structure may be different portionsof one larger RNA molecule, or they may be separate RNA molecules. Wherethe two strands are part of one larger molecule, and therefore areconnected by an uninterrupted chain of nucleotides between the 3′-end ofone strand and the 5′-end of the respective other strand forming theduplex structure, the connecting RNA chain is referred to as a “hairpinloop.” Where the two strands are connected covalently by means otherthan an uninterrupted chain of nucleotides between the 3′-end of onestrand and the 5′-end of the respective other strand forming the duplexstructure, the connecting structure is referred to as a “linker.” TheRNA strands may have the same or a different number of nucleotides. Themaximum number of base pairs is the number of nucleotides in theshortest strand of the dsRNA minus any overhangs that are present in theduplex. In addition to the duplex structure, a dsRNA may comprise one ormore nucleotide overhangs. The term “siRNA” is also used herein to referto a dsRNA as described above.

As used herein, a “nucleotide overhang” refers to the unpairednucleotide or nucleotides that protrude from the duplex structure of adsRNA when a 3′-end of one strand of the dsRNA extends beyond the 5′-endof the other strand, or vice versa. “Blunt” or “blunt end” means thatthere are no unpaired nucleotides at that end of the dsRNA, i.e., nonucleotide overhang. A “blunt ended” dsRNA is a dsRNA that isdouble-stranded over its entire length, i.e., no nucleotide overhang ateither end of the molecule.

The term “antisense strand” refers to the strand of a dsRNA whichincludes a region that is complementary, e.g., fully complementary orsubstantially complementary to a target sequence. As used herein, theterm “region of complementarity” refers to the region on the antisensestrand that is complementary to a sequence, for example a targetsequence, as defined herein. Where the region of complementarity is notfully complementary to the target sequence, the mismatches are mosttolerated in the terminal regions and, if present, are generally in aterminal region or regions, e.g., within 6, 5, 4, 3, or 2 nucleotides ofthe 5′ and/or 3′ terminus.

The term “sense strand,” as used herein, refers to the strand of a dsRNAthat includes a region that is complementary, e.g., fully orsubstantially complementary to a region of the antisense strand.

The term “start position” refers to a nucleotide position on the targetmRNA where the 5′ most nucleotide of a dsRNA sense strand aligns withthe nucleotide position on the target mRNA. For example, a dsRNA with astart position of 382 on NM_021175.2 (SEQ ID NO:1) would includeAD-11459 because position 382 on NM_021175.2 (SEQ ID NO:1) is G and thesense sequence of AD-11459 is 5′-GAAcAuAGGucuuGGAAuAdTsdT-3′ (SEQ IDNO:−30), where G is the 5′ most nucleotide of the sense strand ofAD-11459; thus G at position 382 on NM_021175.2 (SEQ ID NO:1) is thestart position of AD-11459.

As used herein, the term “nucleic acid lipid particle” includes the term“SNALP” and refers to a vesicle of lipids coating a reduced aqueousinterior comprising a nucleic acid such as a dsRNA or a plasmid fromwhich a dsRNA is transcribed. Nucleic acid lipid particles, e.g., SNALPare described, e.g., in U.S. Patent Application Publication Nos.20060240093, 20070135372, and U.S. Ser. No. 61/045,228 filed on Apr. 15,2008. These applications are hereby incorporated by reference.

“Introducing into a cell,” when referring to a dsRNA, means facilitatinguptake or absorption into the cell, as is understood by those skilled inthe art. Absorption or uptake of dsRNA can occur through unaideddiffusive or active cellular processes, or by auxiliary agents ordevices. The meaning of this term is not limited to cells in vitro; adsRNA may also be “introduced into a cell,” wherein the cell is part ofa living organism. In such instance, introduction into the cell willinclude the delivery to the organism. For example, for in vivo delivery,dsRNA can be injected into a tissue site or administered systemically.In vitro introduction into a cell includes methods known in the art suchas electroporation and lipofection. Further approaches are describedherein or known in the art.

The terms “silence,” “inhibit the expression of,” “down-regulate theexpression of,” “suppress the expression of” and the like in as far asthey refer to a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/orNEO1 gene, herein refer to the at least partial suppression of theexpression of a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/orNEO1 gene, as manifested by a reduction of the amount of mRNA which maybe isolated from a first cell or group of cells in which a HAMP, HFE2,HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene is transcribedand which has or have been treated such that the expression of a HAMP,HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene isinhibited, as compared to a second cell or group of cells substantiallyidentical to the first cell or group of cells but which has or have notbeen so treated (control cells). The degree of inhibition is usuallyexpressed in terms of

${\frac{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right) - \left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {treated}\mspace{14mu} {cells}} \right)}{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right)} \cdot 100}\%$

Alternatively, the degree of inhibition may be given in terms of areduction of a parameter that is functionally linked to HAMP, HFE2, HFE,TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene expression, e.g., theamount of protein encoded by a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4,IL6R, BMP6, and/or NEO1 gene which is secreted by a cell, or the numberof cells displaying a certain phenotype, e.g., apoptosis. In principle,HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 genesilencing may be determined in any cell expressing the target, eitherconstitutively or by genomic engineering, and by any appropriate assay.However, when a reference is needed in order to determine whether agiven dsRNA inhibits the expression of a HAMP, HFE2, HFE, TFR2, BMPR1a,SMAD4, IL6R, BMP6, and/or NEO1 gene by a certain degree and therefore isencompassed by the instant invention, the assays provided in theExamples below shall serve as such reference.

For example, in certain instances, expression of a HAMP, HFE2, HFE,TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene is suppressed by atleast about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% byadministration of the double-stranded oligonucleotide featured in theinvention. In some embodiments, a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4,IL6R, BMP6, and/or NEO1 gene is suppressed by at least about 60%, 70%,or 80% by administration of the double-stranded oligonucleotide featuredin the invention. In some embodiments, a HAMP, HFE2, HFE, TFR2, BMPR1a,SMAD4, IL6R, BMP6, and/or NEO1 gene is suppressed by at least about 85%,90%, or 95% by administration of the double-stranded oligonucleotidefeatured in the invention.

As used herein in the context of HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4,IL6R, BMP6, and/or NEO1 expression, the terms “treat,” “treatment,” andthe like, refer to relief from or alleviation of pathological processesmediated by HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/orNEO1 expression. In the context of the present invention insofar as itrelates to any of the other conditions recited herein below (other thanpathological processes mediated by HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4,IL6R, BMP6, and/or NEO1 expression), the terms “treat,” “treatment,” andthe like mean to relieve or alleviate at least one symptom associatedwith such condition, or to slow or reverse the progression of suchcondition.

As used herein, the phrases “effective amount” refers to an amount thatprovides a benefit in the treatment, prevention, or management ofpathological processes mediated by HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4,IL6R, BMP6, and/or NEO1 expression or an overt symptom of pathologicalprocesses mediated by HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6,and/or NEO1 expression. The specific amount that is effective can bereadily determined by an ordinary medical practitioner, and may varydepending on factors known in the art, such as, for example, the type ofpathological processes mediated by HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4,IL6R, BMP6, and/or NEO1 expression, the patient's history and age, thestage of pathological processes mediated by HAMP, HFE2, HFE, TFR2,BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 expression, and theadministration of other anti-pathological processes mediated by HAMP,HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 expressionagents.

As used herein, a “pharmaceutical composition” comprises apharmacologically effective amount of a dsRNA and a pharmaceuticallyacceptable carrier. As used herein, “pharmacologically effectiveamount,” or simply “effective amount” refers to that amount of an RNAeffective to produce the intended pharmacological, therapeutic orpreventive result. For example, if a given clinical treatment isconsidered effective when there is at least a 25% reduction in ameasurable parameter associated with a disease or disorder, apharmacologically effective amount of a drug for the treatment of thatdisease or disorder is the amount necessary to effect at least a 25%reduction in that parameter. For example, a pharmacologically effectiveamount of a dsRNA targeting HAMP can reduce HAMP serum levels by atleast 25%.

The term “pharmaceutically acceptable carrier” refers to a carrier foradministration of a therapeutic agent. Such carriers include, but arenot limited to, saline, buffered saline, dextrose, water, glycerol,ethanol, and combinations thereof. The term specifically excludes cellculture medium. For drugs administered orally, pharmaceuticallyacceptable carriers include, but are not limited to pharmaceuticallyacceptable excipients such as inert diluents, disintegrating agents,binding agents, lubricating agents, sweetening agents, flavoring agents,coloring agents and preservatives. Suitable inert diluents includesodium and calcium carbonate, sodium and calcium phosphate, and lactose,while corn starch and alginic acid are suitable disintegrating agents.Binding agents may include starch and gelatin, while the lubricatingagent, if present, will generally be magnesium stearate, stearic acid ortalc. If desired, the tablets may be coated with a material such asglyceryl monostearate or glyceryl distearate, to delay absorption in thegastrointestinal tract.

As used herein, a “transformed cell” is a cell into which a vector hasbeen introduced from which a dsRNA molecule may be expressed.

Double-Stranded Ribonucleic Acid (dsRNA)

As described in more detail herein, the invention providesdouble-stranded ribonucleic acid (dsRNA) molecules for inhibiting theexpression of a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/orNEO1 gene in a cell or mammal, where the dsRNA includes an antisensestrand having a region of complementarity which is complementary to atleast a part of an mRNA formed in the expression of a HAMP, HFE2, HFE,TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene, and where the regionof complementarity is less than 30 nucleotides in length, generally19-24 nucleotides in length, and where said dsRNA, upon contact with acell expressing said HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6,and/or NEO1 gene, inhibits the expression of said HAMP, HFE2, HFE, TFR2,BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene by at least 30% as assayedby, for example, a PCR or branched DNA (bDNA)-based method, or by aprotein-based method, such as by Western blot. Expression of a HAMP,HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene can bereduced by at least 30% when measured by an assay as described in theExamples below. For example, expression of a HAMP gene in cell culture,such as in Hep3B cells, can be assayed by measuring HAMP mRNA levels,such as by bDNA or TaqMan assay, or by measuring protein levels, such asby ELISA assay. The dsRNA of the invention can further include one ormore single-stranded nucleotide overhangs.

The dsRNA can be synthesized by standard methods known in the art asfurther discussed below, e.g., by use of an automated DNA synthesizer,such as are commercially available from, for example, Biosearch, AppliedBiosystems, Inc. The dsRNA includes two RNA strands that aresufficiently complementary to hybridize to form a duplex structure. Onestrand of the dsRNA (the antisense strand) includes a region ofcomplementarity that is substantially complementary, and generally fullycomplementary, to a target sequence, derived from the sequence of anmRNA formed during the expression of a HAMP, HFE2, HFE, TFR2, BMPR1a,SMAD4, IL6R, BMP6, and/or NEO1 gene, the other strand (the sense strand)includes a region that is complementary to the antisense strand, suchthat the two strands hybridize and form a duplex structure when combinedunder suitable conditions. Generally, the duplex structure is between 15and 30 or between 25 and 30, or between 18 and 25, or between 19 and 24,or between 19 and 21, or 19, 20, or 21 base pairs in length. In oneembodiment the duplex is 19 base pairs in length. In another embodimentthe duplex is 21 base pairs in length. When two different dsRNAs areused in combination, the duplex lengths can be identical or can differ.In one embodiment, the antisense strand of the dsRNA is sufficientlycomplementary to a target mRNA (e.g., a HAMP, HFE2, HFE, TFR2, BMPR1a,SMAD4, IL6R, BMP6, and/or NEO1 mRNA) so as to cause cleavage of thetarget mRNA.

Each strand of the dsRNA of invention is generally between 15 and 30, orbetween 18 and 25, or 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides inlength. In other embodiments, each is strand is 25-30 nucleotides inlength. Each strand of the duplex can be the same length or of differentlengths. When two different siRNAs are used in combination, the lengthsof each strand of each siRNA can be identical or can differ.

The dsRNA of the invention can include one or more single-strandedoverhang(s) of one or more nucleotides. In one embodiment, at least oneend of the dsRNA has a single-stranded nucleotide overhang of 1 to 4,generally 1 or 2 nucleotides. In another embodiment, the antisensestrand of the dsRNA has 1-10 nucleotides overhangs each at the 3′ endand the 5′ end over the sense strand. In further embodiments, the sensestrand of the dsRNA has 1-10 nucleotides overhangs each at the 3′ endand the 5′ end over the antisense strand. The dsRNA can include a 3′overhang of 2 nucleotides on both the sense and antisense strands.

A dsRNAs having at least one nucleotide overhang can have unexpectedlysuperior inhibitory properties than the blunt-ended counterpart. In someembodiments the presence of only one nucleotide overhang strengthens theinterference activity of the dsRNA, without affecting its overallstability. A dsRNA having only one overhang has proven particularlystable and effective in vivo, as well as in a variety of cells, cellculture mediums, blood, and serum. Generally, the single-strandedoverhang is located at the 3′-terminal end of the antisense strand or,alternatively, at the 3′-terminal end of the sense strand. The dsRNA canalso have a blunt end, generally located at the 5′-end of the antisensestrand. Such dsRNAs can have improved stability and inhibitory activity,thus allowing administration at low dosages, i.e., less than 5 mg/kgbody weight of the recipient per day. Generally, the antisense strand ofthe dsRNA has a nucleotide overhang at the 3′-end, and the 5′-end isblunt. In another embodiment, one or more of the nucleotides in theoverhang is replaced with a nucleoside thiophosphate.

In one embodiment, a HAMP gene is a human HAMP gene. In specificembodiments, the sense strand of the dsRNA is one of the sense sequencesfrom Tables 2, 3, 4, and 5, and the antisense strand is one of theantisense sequences of Tables 2, 3, 4, and 5. Alternative antisenseagents that target elsewhere in the target sequence provided in Tables2, 3, 4, and 5 can readily be determined using the target sequence andthe flanking HAMP sequence.

The skilled person is well aware that dsRNAs having a duplex structureof between 20 and 23, but specifically 21, base pairs have been hailedas particularly effective in inducing RNA interference (Elbashir et al.,EMBO 2001, 20:6877-6888). However, others have found that shorter orlonger dsRNAs can be effective as well. In the embodiments describedabove, by virtue of the nature of the oligonucleotide sequences providedin Tables 2, 3, 4, and 5, the dsRNAs featured in the invention caninclude at least one strand of a length described herein. It can bereasonably expected that shorter dsRNAs having one of the sequences ofTables 2, 3, 4, and 5 minus only a few nucleotides on one or both endsmay be similarly effective as compared to the dsRNAs described above.Hence, dsRNAs having a partial sequence of at least 15, 16, 17, 18, 19,20, or more contiguous nucleotides from one of the sequences of Tables2, 3, 4, and 5, and differing in their ability to inhibit the expressionof a HAMP gene in an assay as described herein below by not more than 5,10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the fullsequence, are contemplated by the invention. Further, dsRNAs that cleavewithin a desired HAMP target sequence can readily be made using thecorresponding HAMP antisense sequence and a complementary sensesequence.

In addition, the dsRNAs provided in Tables 2, 3, 4, and 5 identify asite in a HAMP that is susceptible to RNAi based cleavage. As such, thepresent invention further features dsRNAs that target within thesequence targeted by one of the agents of the present invention. As usedherein, a second dsRNA is said to target within the sequence of a firstdsRNA if the second dsRNA cleaves the message anywhere within the mRNAthat is complementary to the antisense strand of the first dsRNA. Such asecond dsRNA will generally consist of at least 15 contiguousnucleotides from one of the sequences provided in Tables 2, 3, 4, and 5coupled to additional nucleotide sequences taken from the regioncontiguous to the selected sequence in a HAMP gene.

In one embodiment, a HFE2 gene is a human HFE2 gene. In specificembodiments, the sense strand of the dsRNA is one of the sense sequencesfrom Table 10A, and the antisense strand is one of the antisensesequences of Table 10A. Alternative antisense agents that targetelsewhere in the target sequence provided in Table 10A can readily bedetermined using the target sequence and the flanking HFE2 sequence.

In the embodiments described above, by virtue of the nature of theoligonucleotide sequences provided in Table 10A, the dsRNAs featured inthe invention can include at least one strand of a length describedherein. It can be reasonably expected that shorter dsRNAs having one ofthe sequences of Table 10A minus only a few nucleotides on one or bothends may be similarly effective as compared to the dsRNAs describedabove. Hence, dsRNAs having a partial sequence of at least 15, 16, 17,18, 19, 20, or more contiguous nucleotides from one of the sequences ofTable 10A, and differing in their ability to inhibit the expression of aHFE2 gene in an assay as described herein below by not more than 5, 10,15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence,are contemplated by the invention. Further, dsRNAs that cleave within adesired HFE2 target sequence can readily be made using the correspondingHFE2 antisense sequence and a complementary sense sequence.

In addition, the dsRNAs provided in Table 10A identify a site in a HFE2that is susceptible to RNAi based cleavage. As such, the presentinvention further features dsRNAs that target within the sequencetargeted by one of the agents of the present invention. As used herein,a second dsRNA is said to target within the sequence of a first dsRNA ifthe second dsRNA cleaves the message anywhere within the mRNA that iscomplementary to the antisense strand of the first dsRNA. Such a seconddsRNA will generally consist of at least 15 contiguous nucleotides fromone of the sequences provided in Table 10A coupled to additionalnucleotide sequences taken from the region contiguous to the selectedsequence in a HFE2 gene.

In one embodiment, a TFR2 gene is a human TFR2 gene. In specificembodiments, the sense strand of the dsRNA is one of the sense sequencesfrom Table 10B or 13, and the antisense strand is one of the antisensesequences of Table 10B or 13. Alternative antisense agents that targetelsewhere in the target sequence provided in Table 10B or 13 can readilybe determined using the target sequence and the flanking TFR2 sequence.

In the embodiments described above, by virtue of the nature of theoligonucleotide sequences provided in Table 10B or 13, the dsRNAsfeatured in the invention can include at least one strand of a lengthdescribed herein. It can be reasonably expected that shorter dsRNAshaving one of the sequences of Table 10B or 13 minus only a fewnucleotides on one or both ends may be similarly effective as comparedto the dsRNAs described above. Hence, dsRNAs having a partial sequenceof at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides fromone of the sequences of Table 10B or 13, and differing in their abilityto inhibit the expression of a TFR2 gene in an assay as described hereinbelow by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNAcomprising the full sequence, are contemplated by the invention.Further, dsRNAs that cleave within a desired TFR2 target sequence canreadily be made using the corresponding TFR2 antisense sequence and acomplementary sense sequence.

In addition, the dsRNAs provided in Table 10B or 13 identify a site in aTFR2 that is susceptible to RNAi based cleavage. As such, the presentinvention further features dsRNAs that target within the sequencetargeted by one of the agents of the present invention. As used herein,a second dsRNA is said to target within the sequence of a first dsRNA ifthe second dsRNA cleaves the message anywhere within the mRNA that iscomplementary to the antisense strand of the first dsRNA. Such a seconddsRNA will generally consist of at least 15 contiguous nucleotides fromone of the sequences provided in Table 10B or 13 coupled to additionalnucleotide sequences taken from the region contiguous to the selectedsequence in a TFR2 gene.

In one embodiment, a SMAD4 gene is a human SMAD4 gene. In specificembodiments, the sense strand of the dsRNA is one of the sense sequencesfrom Table 15 or 16, and the antisense strand is one of the antisensesequences of Table 15 or 16. Alternative antisense agents that targetelsewhere in the target sequence provided in Table 15 or 16 can readilybe determined using the target sequence and the flanking SMAD4 sequence.

In the embodiments described above, by virtue of the nature of theoligonucleotide sequences provided in Table 15 or 16, the dsRNAsfeatured in the invention can include at least one strand of a lengthdescribed herein. It can be reasonably expected that shorter dsRNAshaving one of the sequences of Table 15 or 16 minus only a fewnucleotides on one or both ends may be similarly effective as comparedto the dsRNAs described above. Hence, dsRNAs having a partial sequenceof at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides fromone of the sequences of Table 15 or 16, and differing in their abilityto inhibit the expression of a SMAD4 gene in an assay as describedherein below by not more than 5, 10, 15, 20, 25, or 30% inhibition froma dsRNA comprising the full sequence, are contemplated by the invention.Further, dsRNAs that cleave within a desired SMAD4 target sequence canreadily be made using the corresponding SMAD4 antisense sequence and acomplementary sense sequence.

In addition, the dsRNAs provided in Table 15 or 16 identify a site in aSMAD4 that is susceptible to RNAi based cleavage. As such, the presentinvention further features dsRNAs that target within the sequencetargeted by one of the agents of the present invention. As used herein,a second dsRNA is said to target within the sequence of a first dsRNA ifthe second dsRNA cleaves the message anywhere within the mRNA that iscomplementary to the antisense strand of the first dsRNA. Such a seconddsRNA will generally consist of at least 15 contiguous nucleotides fromone of the sequences provided in Table 15 or 16 coupled to additionalnucleotide sequences taken from the region contiguous to the selectedsequence in a SMAD4 gene.

In one embodiment, a NEO1 gene is a human NEO1 gene. In specificembodiments, the sense strand of the dsRNA is one of the sense sequencesfrom Table 17 or 18, and the antisense strand is one of the antisensesequences of Table 17 or 18. Alternative antisense agents that targetelsewhere in the target sequence provided in Table 17 or 18 can readilybe determined using the target sequence and the flanking NEO1 sequence.

In the embodiments described above, by virtue of the nature of theoligonucleotide sequences provided in Table 17 or 18, the dsRNAsfeatured in the invention can include at least one strand of a lengthdescribed herein. It can be reasonably expected that shorter dsRNAshaving one of the sequences of Table 17 or 18 minus only a fewnucleotides on one or both ends may be similarly effective as comparedto the dsRNAs described above. Hence, dsRNAs having a partial sequenceof at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides fromone of the sequences of Table 17 or 18, and differing in their abilityto inhibit the expression of a NEO1 gene in an assay as described hereinbelow by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNAcomprising the full sequence, are contemplated by the invention.Further, dsRNAs that cleave within a desired NEO1 target sequence canreadily be made using the corresponding NEO1 antisense sequence and acomplementary sense sequence.

In addition, the dsRNAs provided in Table 17 or 18 identify a site in aNEO1 that is susceptible to RNAi based cleavage. As such, the presentinvention further features dsRNAs that target within the sequencetargeted by one of the agents of the present invention. As used herein,a second dsRNA is said to target within the sequence of a first dsRNA ifthe second dsRNA cleaves the message anywhere within the mRNA that iscomplementary to the antisense strand of the first dsRNA. Such a seconddsRNA will generally consist of at least 15 contiguous nucleotides fromone of the sequences provided in Table 17 or 18 coupled to additionalnucleotide sequences taken from the region contiguous to the selectedsequence in a NEO1 gene.

In one embodiment, a BMP6 gene is a human BMP6 gene. In specificembodiments, the sense strand of the dsRNA is one of the sense sequencesfrom Table 21, and the antisense strand is one of the antisensesequences of Table 21. Alternative antisense agents that targetelsewhere in the target sequence provided in Table 21 can readily bedetermined using the target sequence and the flanking BMP6 sequence.

In the embodiments described above, by virtue of the nature of theoligonucleotide sequences provided in Table 21, the dsRNAs featured inthe invention can include at least one strand of a length describedherein. It can be reasonably expected that shorter dsRNAs having one ofthe sequences of Table 21 minus only a few nucleotides on one or bothends may be similarly effective as compared to the dsRNAs describedabove. Hence, dsRNAs having a partial sequence of at least 15, 16, 17,18, 19, 20, or more contiguous nucleotides from one of the sequences ofTable 21, and differing in their ability to inhibit the expression of aBMP6 gene in an assay as described herein below by not more than 5, 10,15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence,are contemplated by the invention. Further, dsRNAs that cleave within adesired BMP6 target sequence can readily be made using the correspondingBMP6 antisense sequence and a complementary sense sequence.

In addition, the dsRNAs provided in Table 21 identify a site in a BMP6that is susceptible to RNAi based cleavage. As such, the presentinvention further features dsRNAs that target within the sequencetargeted by one of the agents of the present invention. As used herein,a second dsRNA is said to target within the sequence of a first dsRNA ifthe second dsRNA cleaves the message anywhere within the mRNA that iscomplementary to the antisense strand of the first dsRNA. Such a seconddsRNA will generally consist of at least 15 contiguous nucleotides fromone of the sequences provided in Table 21 coupled to additionalnucleotide sequences taken from the region contiguous to the selectedsequence in a BMP6 gene.

With regard to Tables 4, 10A, 10B, 13, 16, 18, and 21: It should benoted that unmodified versions of each of the modified sequences shownare included within the scope of the invention. “Unmodified version”refers to a sequence that does not include one or more chemicalmodifications, e.g., a 2′-O methyl group, a phosphorothioate, and/or a2′-fluoro group. For example, included in the invention are unmodifiedversions of AD-47391, which targets HFE2. See Table 10A. Unmodifiedsense strand versions of AD-47391 include: AGAGUAGGGAAUCAUGGCUdTdT (SEQID NO: 31) and AGAGUAGGGAAUCAUGGCU (SEQ ID NO: 32). Unmodified antisensestrand versions of AD-47391 include: AGCCAUGAUUCCCUACUCUdTdT (SEQ ID NO:33) and AGCCAUGAUUCCCUACUCU (SEQ ID NO: 34). As another example,included in the invention are unmodified versions of AD-47826, whichtargets TFR2. See Table 10B. Unmodified sense strand versions ofAD-47826 include: CAGGCAGCCAAACCUCAUUdTdT (SEQ ID NO: 35) andCAGGCAGCCAAACCUCAUU (SEQ ID NO: 36). Unmodified antisense strandversions of AD-47826 include: AAUGAGGUUUGGCUGCCUG (SEQ ID NO: 37) andAAUGAGGUUUGGCUGCCUGdTdT (SEQ ID NO: 38).

Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art. The cleavage site on the target mRNA of adsRNA can be determined using methods generally known to one of ordinaryskill in the art, e.g., the 5′-RACE method described in Soutschek etal., Nature; 2004, Vol. 432, pp. 173-178 (which is herein incorporatedby reference for all purposes). Included in the invention are dsRNA thatcleave the RNA target at the same location as the dsRNA described in thetables herein.

The dsRNA featured in the invention can contain one or more mismatchesto the target sequence. In one embodiment, the dsRNA featured in theinvention contains no more than 3 mismatches. If the antisense strand ofthe dsRNA contains mismatches to a target sequence, it is preferablethat the area of mismatch not be located in the center of the region ofcomplementarity. If the antisense strand of the dsRNA containsmismatches to the target sequence, it is preferable that the mismatch berestricted to 5 nucleotides from either end, for example 5, 4, 3, 2, or1 nucleotide from either the 5′ or 3′ end of the region ofcomplementarity. For example, for a 23 nucleotide dsRNA strand which iscomplementary to a region of a HAMP gene, the dsRNA generally does notcontain any mismatch within the central 13 nucleotides. The methodsdescribed within the invention can be used to determine whether a dsRNAcontaining a mismatch to a target sequence is effective in inhibitingthe expression of a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6,and/or NEO1 gene. Consideration of the efficacy of dsRNAs withmismatches in inhibiting expression of a HAMP, HFE2, HFE, TFR2, BMPR1a,SMAD4, IL6R, BMP6, and/or NEO1 gene is important, especially if theparticular region of complementarity in a HAMP, HFE2, HFE, TFR2, BMPR1a,SMAD4, IL6R, BMP6, and/or NEO1 gene is known to have polymorphicsequence variation within the population.

Modifications

In yet another embodiment, the dsRNA is chemically modified to enhancestability. The nucleic acids featured in the invention may besynthesized and/or modified by methods well established in the art, suchas those described in “Current protocols in nucleic acid chemistry,”Beaucage, S. L. et al. (Eds.), John Wiley & Sons, Inc., New York, N.Y.,USA, which is hereby incorporated herein by reference. Specific examplesof dsRNA compounds useful in this invention include dsRNAs containingmodified backbones or no natural internucleoside linkages. As defined inthis specification, dsRNAs having modified backbones include those thatretain a phosphorus atom in the backbone and those that do not have aphosphorus atom in the backbone. For the purposes of this specification,and as sometimes referenced in the art, modified dsRNAs that do not havea phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Modified dsRNA backbones include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those) having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799;5,587,361; and 5,625,050, each of which is herein incorporated byreference

Modified dsRNA backbones that do not include a phosphorus atom thereinhave backbones that are formed by short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatoms and alkyl or cycloalkylinternucleoside linkages, or ore or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts. In some instances, dsRNAs can be made with“Light Fluoro” chemical modifications as follows: all pyrimidines(cytosine and uridine) in the sense strand can be replaced withcorresponding 2′-Fluoro bases (2′ Fluoro C and 2′-Fluoro U). In theantisense strand, pyrimidines adjacent to (towards 5′ position) ribo Anucleoside can be replaced with their corresponding 2-Fluoronucleosides.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and,5,677,439, each of which is herein incorporated by reference.

In other suitable dsRNA mimetics, both the sugar and the internucleosidelinkage, i.e., the backbone, of the nucleotide units are replaced withnovel groups. The base units are maintained for hybridization with anappropriate nucleic acid target compound. One such oligomeric compound,a dsRNA mimetic that has been shown to have excellent hybridizationproperties, is referred to as a peptide nucleic acid (PNA). In PNAcompounds, the sugar backbone of a dsRNA is replaced with an amidecontaining backbone, in particular an aminoethylglycine backbone. Thenucleobases are retained and are bound directly or indirectly to azanitrogen atoms of the amide portion of the backbone. Representative U.S.patents that teach the preparation of PNA compounds include, but are notlimited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each ofwhich is herein incorporated by reference. Further teaching of PNAcompounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

Other embodiments of the invention are dsRNAs with phosphorothioatebackbones and oligonucleosides with heteroatom backbones, and inparticular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known as a methylene(methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of theabove-referenced U.S. Pat. No. 5,489,677, and the amide backbones of theabove-referenced U.S. Pat. No. 5,602,240. Also preferred are dsRNAshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modified dsRNAs may also contain one or more substituted sugar moieties.Preferred dsRNAs comprise one of the following at the 2′ position: OH;F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; orO-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may besubstituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl andalkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.Other preferred dsRNAs comprise one of the following at the 2′ position:C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl,O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃,SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of an dsRNA, or a group for improving thepharmacodynamic properties of an dsRNA, and other substituents havingsimilar properties. A preferred modification includes 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martinet al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxy-alkoxygroup. A further preferred modification includes2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as2′-DMAOE, as described in examples herein below, and2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples herein below.

Other preferred modifications include 2′-methoxy (2′-OCH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on the dsRNA,particularly the 3′ position of the sugar on the 3′ terminal nucleotideor in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide.DsRNAs may also have sugar mimetics such as cyclobutyl moieties in placeof the pentofuranosyl sugar. Representative U.S. patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which arecommonly owned with the instant application, and each of which is hereinincorporated by reference in its entirety.

dsRNAs may also include nucleobase (often referred to in the art simplyas “base”) modifications or substitutions. As used herein, “unmodified”or “natural” nucleobases include the purine bases adenine (A) andguanine (G), and the pyrimidine bases thymine (T), cytosine (C) anduracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substitutedadenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyland other 5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons,1990, these disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, YS., Chapter 15, DsRNA Research and Applications, pages 289-302, Crooke,S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobasesare particularly useful for increasing the binding affinity of theoligomeric compounds featured in the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., Eds., DsRNA Research and Applications,CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. No.3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066;5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091;5,614,617; and 5,681,941, each of which is herein incorporated byreference, and U.S. Pat. No. 5,750,692, also herein incorporated byreference.

Conjugates

Another modification of the dsRNAs of the invention involves chemicallylinking to the dsRNA one or more moieties or conjugates which enhancethe activity, cellular distribution or cellular uptake of the dsRNA.Such moieties include but are not limited to lipid moieties such as acholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989,86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let.,1994, 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan etal., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg.Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser etal., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g.,dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991,10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuket al., Biochimie, 1993, 75:49-54), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-Hphosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res.,1990, 18:3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264:229-237), or an octadecylamine orhexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923-937).

In some embodiments of the compositions and methods of the invention, andsRNA oligonucleotide further comprises a carbohydrate. The carbohydrateconjugated dsRNA are advantageous for the in vivo delivery of nucleicacids, as well as compositions suitable for in vivo therapeutic use, asdescribed herein. As used herein, “carbohydrate” refers to a compoundwhich is either a carbohydrate per se made up of one or moremonosaccharide units having at least 6 carbon atoms (which can belinear, branched or cyclic) with an oxygen, nitrogen or sulfur atombonded to each carbon atom; or a compound having as a part thereof acarbohydrate moiety made up of one or more monosaccharide units eachhaving at least six carbon atoms (which can be linear, branched orcyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbonatom. Representative carbohydrates include the sugars (mono-, di-, tri-and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9monosaccharide units), and polysaccharides such as starches, glycogen,cellulose and polysaccharide gums. Specific monosaccharides include C5and above (e.g., C5, C6, C7, or C8) sugars; di- and trisaccharidesinclude sugars having two or three monosaccharide units (e.g., C5, C6,C7, or C8).

In one embodiment, a carbohydrate conjugate for use in the compositionsand methods of the invention is a monosaccharide. In one embodiment, themonosaccharide is an N-acetylgalactosamine, such as

In another embodiment, a carbohydrate conjugate for use in thecompositions and methods of the invention is selected from the groupconsisting of:

Another representative carbohydrate conjugate for use in the embodimentsdescribed herein includes, but is not limited to,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

In some embodiments, the carbohydrate conjugate further comprises one ormore additional ligands as described above, such as, but not limited to,a PK modulator and/or a cell permeation peptide.

In some embodiments, the conjugate or ligand described herein can beattached to an dsRNA oligonucleotide with various linkers that can becleavable or non cleavable.

The term “linker” or “linking group” means an organic moiety thatconnects two parts of a compound, e.g., covalently attaches two parts ofa compound. Linkers typically comprise a direct bond or an atom such asoxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO₂, SO₂NH or achain of atoms, such as, but not limited to, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl,heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl,heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl,heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl,alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl,alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl,alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl,alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl,alkenylheteroarylalkenyl, alkenylheteroarylalkynyl,alkynylheteroarylalkyl, alkynylheteroarylalkenyl,alkynylheteroarylalkynyl, alkylheterocyclylalkyl,alkylheterocyclylalkenyl, alkylhererocyclylalkynyl,alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl,alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl,alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl,alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl,alkynylhereroaryl, which one or more methylenes can be interrupted orterminated by O, S, S(O), SO₂, N(R8), C(O), substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic orsubstituted aliphatic. In one embodiment, the linker is between about1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17,8-17, 6-16, 7-17, or 8-16 atoms.

A cleavable linking group is one which is sufficiently stable outsidethe cell, but which upon entry into a target cell is cleaved to releasethe two parts the linker is holding together. In a preferred embodiment,the cleavable linking group is cleaved at least about 10 times, 20,times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90times or more, or at least about 100 times faster in a target cell orunder a first reference condition (which can, e.g., be selected to mimicor represent intracellular conditions) than in the blood of a subject,or under a second reference condition (which can, e.g., be selected tomimic or represent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH,redox potential or the presence of degradative molecules. Generally,cleavage agents are more prevalent or found at higher levels oractivities inside cells than in serum or blood. Examples of suchdegradative agents include: redox agents which are selected forparticular substrates or which have no substrate specificity, including,e.g., oxidative or reductive enzymes or reductive agents such asmercaptans, present in cells, that can degrade a redox cleavable linkinggroup by reduction; esterases; endosomes or agents that can create anacidic environment, e.g., those that result in a pH of five or lower;enzymes that can hydrolyze or degrade an acid cleavable linking group byacting as a general acid, peptidases (which can be substrate specific),and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptibleto pH. The pH of human serum is 7.4, while the average intracellular pHis slightly lower, ranging from about 7.1-7.3. Endosomes have a moreacidic pH, in the range of 5.5-6.0, and lysosomes have an even moreacidic pH at around 5.0. Some linkers will have a cleavable linkinggroup that is cleaved at a preferred pH, thereby releasing a cationiclipid from the ligand inside the cell, or into the desired compartmentof the cell.

A linker can include a cleavable linking group that is cleavable by aparticular enzyme. The type of cleavable linking group incorporated intoa linker can depend on the cell to be targeted. For example, aliver-targeting ligand can be linked to a cationic lipid through alinker that includes an ester group. Liver cells are rich in esterases,and therefore the linker will be cleaved more efficiently in liver cellsthan in cell types that are not esterase-rich. Other cell-types rich inesterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell typesrich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group canbe evaluated by testing the ability of a degradative agent (orcondition) to cleave the candidate linking group. It will also bedesirable to also test the candidate cleavable linking group for theability to resist cleavage in the blood or when in contact with othernon-target tissue. Thus, one can determine the relative susceptibilityto cleavage between a first and a second condition, where the first isselected to be indicative of cleavage in a target cell and the second isselected to be indicative of cleavage in other tissues or biologicalfluids, e.g., blood or serum. The evaluations can be carried out in cellfree systems, in cells, in cell culture, in organ or tissue culture, orin whole animals. It can be useful to make initial evaluations incell-free or culture conditions and to confirm by further evaluations inwhole animals. In preferred embodiments, useful candidate compounds arecleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, orabout 100 times faster in the cell (or under in vitro conditionsselected to mimic intracellular conditions) as compared to blood orserum (or under in vitro conditions selected to mimic extracellularconditions).

In one embodiment, a cleavable linking group is a redox cleavablelinking group that is cleaved upon reduction or oxidation. An example ofreductively cleavable linking group is a disulphide linking group(—S—S—). To determine if a candidate cleavable linking group is asuitable “reductively cleavable linking group,” or for example issuitable for use with a particular dsRNA moiety and particular targetingagent one can look to methods described herein. For example, a candidatecan be evaluated by incubation with dithiothreitol (DTT), or otherreducing agent using reagents know in the art, which mimic the rate ofcleavage which would be observed in a cell, e.g., a target cell. Thecandidates can also be evaluated under conditions which are selected tomimic blood or serum conditions. In one, candidate compounds are cleavedby at most about 10% in the blood. In other embodiments, usefulcandidate compounds are degraded at least about 2, 4, 10, 20, 30, 40,50, 60, 70, 80, 90, or about 100 times faster in the cell (or under invitro conditions selected to mimic intracellular conditions) as comparedto blood (or under in vitro conditions selected to mimic extracellularconditions). The rate of cleavage of candidate compounds can bedetermined using standard enzyme kinetics assays under conditions chosento mimic intracellular media and compared to conditions chosen to mimicextracellular media.

In another embodiment, a cleavable linker comprises a phosphate-basedcleavable linking group. A phosphate-based cleavable linking group iscleaved by agents that degrade or hydrolyze the phosphate group. Anexample of an agent that cleaves phosphate groups in cells are enzymessuch as phosphatases in cells. Examples of phosphate-based linkinggroups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—,—S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—,—S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—,—S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—. Preferred embodimentsare —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—,—O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—,—O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O—, —S—P(S)(H)—O—,—S—P(O)(H)—S—, —O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—.These candidates can be evaluated using methods analogous to thosedescribed above.

In another embodiment, a cleavable linker comprises an acid cleavablelinking group. An acid cleavable linking group is a linking group thatis cleaved under acidic conditions. In preferred embodiments acidcleavable linking groups are cleaved in an acidic environment with a pHof about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower),or by agents such as enzymes that can act as a general acid. In a cell,specific low pH organelles, such as endosomes and lysosomes can providea cleaving environment for acid cleavable linking groups. Examples ofacid cleavable linking groups include but are not limited to hydrazones,esters, and esters of amino acids. Acid cleavable groups can have thegeneral formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is whenthe carbon attached to the oxygen of the ester (the alkoxy group) is anaryl group, substituted alkyl group, or tertiary alkyl group such asdimethyl pentyl or t-butyl. These candidates can be evaluated usingmethods analogous to those described above.

In another embodiment, a cleavable linker comprises an ester-basedcleavable linking group. An ester-based cleavable linking group iscleaved by enzymes such as esterases and amidases in cells. Examples ofester-based cleavable linking groups include but are not limited toesters of alkylene, alkenylene and alkynylene groups. Ester cleavablelinking groups have the general formula —C(O)O—, or —OC(O)—. Thesecandidates can be evaluated using methods analogous to those describedabove.

In yet another embodiment, a cleavable linker comprises a peptide-basedcleavable linking group. A peptide-based cleavable linking group iscleaved by enzymes such as peptidases and proteases in cells.Peptide-based cleavable linking groups are peptide bonds formed betweenamino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.)and polypeptides. Peptide-based cleavable groups do not include theamide group (—C(O)NH—). The amide group can be formed between anyalkylene, alkenylene or alkynelene. A peptide bond is a special type ofamide bond formed between amino acids to yield peptides and proteins.The peptide based cleavage group is generally limited to the peptidebond (i.e., the amide bond) formed between amino acids yielding peptidesand proteins and does not include the entire amide functional group.Peptide-based cleavable linking groups have the general formula—NHCHRAC(O)NHCHRBC(O)—, where RA and RB are the R groups of the twoadjacent amino acids. These candidates can be evaluated using methodsanalogous to those described above.

In one embodiment, an dsRNA of the invention is conjugated to acarbohydrate through a linker. Non-limiting examples of dsRNAcarbohydrate conjugates with linkers of the compositions and methods ofthe invention include, but are not limited to,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

In certain embodiments of the compositions and methods of the invention,a ligand is one or more GalNAc (N-acetylgalactosamine) derivativesattached through a bivalent or trivalent branched linker.

In one embodiment, a dsRNA of the invention is conjugated to a bivalentor trivalent branched linker selected from the group of structures shownin any of formula (XXXI)-(XXXIV):

wherein:

q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independentlyfor each occurrence 0-20 and wherein the repeating unit can be the sameor different;

P^(2A), P^(2B), P^(3A), P^(3B), P^(4A), P^(4B), P^(5A), P^(5B), P^(5C),T^(2A), T^(2B), T^(3A), T^(3B), T^(4A), T^(4B), T^(4A), T^(5B), T^(5C)are each independently for each occurrence absent, CO, NH, O, S, OC(O),NHC(O), CH₂, CH₂NH or CH₂O;

Q^(2A), Q^(2B), Q^(3A), Q^(3B), Q^(4A), Q^(4B), Q^(5A), Q^(5B), Q^(5C)are independently for each occurrence absent, alkylene, substitutedalkylene wherein one or more methylenes can be interrupted or terminatedby one or more of O, S, S(O), SO₂, N(R^(N)), C(R′)═C(R″), C≡C or C(O);

R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), R^(5A), R^(5B), R^(5C)are each independently for each occurrence absent, NH, O, S, CH₂, C(O)O,C(O)NH, NHCH(R^(a))C(O), —C(O)—CH(R^(a))—NH—, CO, CH═N—O,

or heterocyclyl;

L^(2A), L^(2B), L^(3A), L^(3B), L^(4A), L^(4B), L^(5A), L^(5B), L^(5C)represent the ligand; i.e. each independently for each occurrence amonosaccharide (such as GalNAc), disaccharide, trisaccharide,tetrasaccharide, oligosaccharide, or polysaccharide; and R^(a) is H oramino acid side chain. Trivalent conjugating GalNAc derivatives areparticularly useful for use with RNAi agents for inhibiting theexpression of a target gene, such as those of formula (XXXV):

wherein L^(5A), L^(5B) and L^(5C) represent a monosaccharide, such asGalNAc derivative.

Examples of suitable bivalent and trivalent branched linker groupsconjugating GalNAc derivatives include, but are not limited to, thestructures recited above as formulas II_VII, XI, X, and XIII.

Representative U.S. patents that teach the preparation of RNA conjugatesinclude, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882;5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203,5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931;6,900,297; 7,037,646; 8,106,022, the entire contents of each of whichare hereby incorporated herein by reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within a dsRNA. The present invention also includesdsRNA compounds which are chimeric compounds. “Chimeric” dsRNA compoundsor “chimeras,” in the context of this invention, are dsRNA compounds,particularly dsRNAs, which contain two or more chemically distinctregions, each made up of at least one monomer unit, i.e., a nucleotidein the case of a dsRNA compound. These dsRNAs typically contain at leastone region wherein the dsRNA is modified so as to confer upon the dsRNAincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target nucleic acid. Anadditional region of the dsRNA may serve as a substrate for enzymescapable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNaseH is a cellular endonuclease which cleaves the RNA strand of an RNA:DNAduplex. Activation of RNase H, therefore, results in cleavage of the RNAtarget, thereby greatly enhancing the efficiency of dsRNA inhibition ofgene expression. Consequently, comparable results can often be obtainedwith shorter dsRNAs when chimeric dsRNAs are used, compared tophosphorothioate deoxydsRNAs hybridizing to the same target region.

In certain instances, the dsRNA may be modified by a non-ligand group. Anumber of non-ligand molecules have been conjugated to dsRNAs in orderto enhance the activity, cellular distribution or cellular uptake of thedsRNA, and procedures for performing such conjugations are available inthe scientific literature. Such non-ligand moieties have included lipidmoieties, such as cholesterol (Letsinger et al., Proc. Natl. Acad. Sci.USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem.Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharanet al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg.Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al.,Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiolor undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111;Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie,1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl.Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264:229), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923). Representative United States patents thatteach the preparation of such dsRNA conjugates have been listed above.Typical conjugation protocols involve the synthesis of dsRNAs bearing anaminolinker at one or more positions of the sequence. The amino group isthen reacted with the molecule being conjugated using appropriatecoupling or activating reagents. The conjugation reaction may beperformed either with the dsRNA still bound to the solid support orfollowing cleavage of the dsRNA in solution phase. Purification of thedsRNA conjugate by HPLC typically affords the pure conjugate.

Vector Encoded dsRNAs

In another aspect, HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6,and/or NEO1 dsRNA molecules are expressed from transcription unitsinserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG.(1996), 12:5-10; Skillern, A., et al., International PCT Publication No.WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, andConrad, U.S. Pat. No. 6,054,299). These transgenes can be introduced asa linear construct, a circular plasmid, or a viral vector, which can beincorporated and inherited as a transgene integrated into the hostgenome. The transgene can also be constructed to permit it to beinherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl.Acad. Sci. USA (1995) 92:1292).

The individual strands of a dsRNA can be transcribed by promoters on twoseparate expression vectors and co-transfected into a target cell.Alternatively each individual strand of the dsRNA can be transcribed bypromoters both of which are located on the same expression plasmid. Inone embodiment, a dsRNA is expressed as an inverted repeat joined by alinker polynucleotide sequence such that the dsRNA has a stem and loopstructure.

The recombinant dsRNA expression vectors are generally DNA plasmids orviral vectors. dsRNA expressing viral vectors can be constructed basedon, but not limited to, adeno-associated virus (for a review, seeMuzyczka, et al., Curr. Topics Micro. Immunol. (1992) 158:97-129));adenovirus (see, for example, Berkner, et al., BioTechniques (1998)6:616), Rosenfeld et al. (1991, Science 252:431-434), and Rosenfeld etal. (1992), Cell 68:143-155)); or alphavirus as well as others known inthe art. Retroviruses have been used to introduce a variety of genesinto many different cell types, including epithelial cells, in vitroand/or in vivo (see, e.g., Eglitis, et al., Science (1985)230:1395-1398; Danos and Mulligan, Proc. Natl. Acad. Sci. USA (1998)85:6460-6464; Wilson et al., 1988, Proc. Natl. Acad. Sci. USA85:3014-3018; Armentano et al., 1990, Proc. Natl. Acad. Sci. USA87:61416145; Huber et al., 1991, Proc. Natl. Acad. Sci. USA88:8039-8043; Ferry et al., 1991, Proc. Natl. Acad. Sci. USA88:8377-8381; Chowdhury et al., 1991, Science 254:1802-1805; vanBeusechem. et al., 1992, Proc. Natl. Acad. Sci. USA 89:7640-19; Kay etal., 1992, Human Gene Therapy 3:641-647; Dai et al., 1992, Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al., 1993, J. Immunol.150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCTApplication WO 89/07136; PCT Application WO 89/02468; PCT Application WO89/05345; and PCT Application WO 92/07573). Recombinant retroviralvectors capable of transducing and expressing genes inserted into thegenome of a cell can be produced by transfecting the recombinantretroviral genome into suitable packaging cell lines such as PA317 andPsi-CRIP (Comette et al., 1991, Human Gene Therapy 2:5-10; Cone et al.,1984, Proc. Natl. Acad. Sci. USA 81:6349). Recombinant adenoviralvectors can be used to infect a wide variety of cells and tissues insusceptible hosts (e.g., rat, hamster, dog, and chimpanzee) (Hsu et al.,1992, J. Infectious Disease, 166:769), and also have the advantage ofnot requiring mitotically active cells for infection.

Any viral vector capable of accepting the coding sequences for the dsRNAmolecule(s) to be expressed can be used, for example vectors derivedfrom adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g.,lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus,and the like. The tropism of viral vectors can be modified bypseudotyping the vectors with envelope proteins or other surfaceantigens from other viruses, or by substituting different viral capsidproteins, as appropriate.

For example, lentiviral vectors featured in the invention can bepseudotyped with surface proteins from vesicular stomatitis virus (VSV),rabies, Ebola, Mokola, and the like. AAV vectors featured in theinvention can be made to target different cells by engineering thevectors to express different capsid protein serotypes. For example, anAAV vector expressing a serotype 2 capsid on a serotype 2 genome iscalled AAV 2/2. This serotype 2 capsid gene in the AAV 2/2 vector can bereplaced by a serotype 5 capsid gene to produce an AAV 2/5 vector.Techniques for constructing AAV vectors which express different capsidprotein serotypes are within the skill in the art; see, e.g., RabinowitzJ E et al. (2002), J Virol 76:791-801, the entire disclosure of which isherein incorporated by reference.

Selection of recombinant viral vectors suitable for use in theinvention, methods for inserting nucleic acid sequences for expressingthe dsRNA into the vector, and methods of delivering the viral vector tothe cells of interest are within the skill in the art. See, for example,Dornburg R (1995), Gene Therap. 2: 301-310; Eglitis M A (1988),Biotechniques 6: 608-614; Miller A D (1990), Hum Gene Therap. 1: 5-14;Anderson W F (1998), Nature 392: 25-30; and Rubinson D A et al., Nat.Genet. 33: 401-406, the entire disclosures of which are hereinincorporated by reference.

Viral vectors can be derived from AV and AAV. In one embodiment, thedsRNA featured in the invention is expressed as two separate,complementary single-stranded RNA molecules from a recombinant AAVvector having, for example, either the U6 or H1 RNA promoters, or thecytomegalovirus (CMV) promoter.

A suitable AV vector for expressing the dsRNA featured in the invention,a method for constructing the recombinant AV vector, and a method fordelivering the vector into target cells, are described in Xia H et al.(2002), Nat. Biotech. 20: 1006-1010.

Suitable AAV vectors for expressing the dsRNA featured in the invention,methods for constructing the recombinant AV vector, and methods fordelivering the vectors into target cells are described in Samulski R etal. (1987), J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol,70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S.Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International PatentApplication No. WO 94/13788; and International Patent Application No. WO93/24641, the entire disclosures of which are herein incorporated byreference.

The promoter driving dsRNA expression in either a DNA plasmid or viralvector featured in the invention may be a eukaryotic RNA polymerase I(e.g., ribosomal RNA promoter), RNA polymerase II (e.g., CMV earlypromoter or actin promoter or U1 snRNA promoter) or generally RNApolymerase III promoter (e.g., U6 snRNA or 7SK RNA promoter) or aprokaryotic promoter, for example the T7 promoter, provided theexpression plasmid also encodes T7 RNA polymerase required fortranscription from a T7 promoter. The promoter can also direct transgeneexpression to the pancreas (see, e.g., the insulin regulatory sequencefor pancreas (Bucchini et al., 1986, Proc. Natl. Acad. Sci. USA83:2511-2515)).

In addition, expression of the transgene can be precisely regulated, forexample, by using an inducible regulatory sequence and expressionsystems such as a regulatory sequence that is sensitive to certainphysiological regulators, e.g., circulating glucose levels, or hormones(Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expressionsystems, suitable for the control of transgene expression in cells or inmammals include regulation by ecdysone, by estrogen, progesterone,tetracycline, chemical inducers of dimerization, andisopropyl-beta-D1-thiogalactopyranoside (EPTG). A person skilled in theart would be able to choose the appropriate regulatory/promoter sequencebased on the intended use of the dsRNA transgene.

Generally, recombinant vectors capable of expressing dsRNA molecules aredelivered as described below, and persist in target cells.Alternatively, viral vectors can be used that provide for transientexpression of dsRNA molecules. Such vectors can be repeatedlyadministered as necessary. Once expressed, the dsRNAs bind to target RNAand modulate its function or expression. Delivery of dsRNA expressingvectors can be systemic, such as by intravenous or intramuscularadministration, by administration to target cells ex-planted from thepatient followed by reintroduction into the patient, or by any othermeans that allows for introduction into a desired target cell.

dsRNA expression DNA plasmids are typically transfected into targetcells as a complex with cationic lipid carriers (e.g., Oligofectamine)or non-cationic lipid-based carriers (e.g., Transit-TKO™). Multiplelipid transfections for dsRNA-mediated knockdowns targeting differentregions of a single HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6,and/or NEO1 gene or multiple HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R,BMP6, and/or NEO1 genes over a period of a week or more are alsocontemplated by the invention. Successful introduction of vectors intohost cells can be monitored using various known methods. For example,transient transfection can be signaled with a reporter, such as afluorescent marker, such as Green Fluorescent Protein (GFP). Stabletransfection of cells ex vivo can be ensured using markers that providethe transfected cell with resistance to specific environmental factors(e.g., antibiotics and drugs), such as hygromycin B resistance.

HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 specificdsRNA molecules can also be inserted into vectors and used as genetherapy vectors for human patients. Gene therapy vectors can bedelivered to a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA91:3054-3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or caninclude a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

Pharmaceutical Compositions Containing dsRNA

In one embodiment, the invention provides pharmaceutical compositionscontaining a dsRNA, as described herein, and a pharmaceuticallyacceptable carrier. The pharmaceutical composition containing the dsRNAis useful for treating a disease or disorder associated with theexpression or activity of a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R,BMP6, and/or NEO1 gene, such as pathological processes mediated by HAMP,HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 expression. Suchpharmaceutical compositions are formulated based on the mode ofdelivery. One example is compositions that are formulated for systemicadministration via parenteral delivery, e.g., by intravenous (IV)delivery. Another example is compositions that are formulated for directdelivery into the brain parenchyma, e.g., by infusion into the brain,such as by continuous pump infusion.

The pharmaceutical compositions featured herein are administered indosages sufficient to inhibit expression of HAMP, HFE2, HFE, TFR2,BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 genes.

In general, a suitable dose of dsRNA will be in the range of 0.01 to200.0 milligrams per kilogram body weight of the recipient per day,generally in the range of 1 to 50 mg per kilogram body weight per day.For example, the dsRNA can be administered at 0.0059 mg/kg, 0.01 mg/kg,0.0295 mg/kg, 0.05 mg/kg, 0.0590 mg/kg, 0.163 mg/kg, 0.2 mg/kg, 0.3mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.543 mg/kg, 0.5900 mg/kg, 0.6 mg/kg, 0.7mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg,1.4 mg/kg, 1.5 mg/kg, 1.628 mg/kg, 2 mg/kg, 3 mg/kg, 5.0 mg/kg, 10mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, or 50 mg/kg per single dose.

In one embodiment, the dosage is between 0.01 and 0.2 mg/kg. Forexample, the dsRNA can be administered at a dose of 0.01 mg/kg, 0.02mg/kg, 0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.06 mg/kg, 0.07 mg/kg 0.08mg/kg 0.09 mg/kg, 0.10 mg/kg, 0.11 mg/kg, 0.12 mg/kg, 0.13 mg/kg, 0.14mg/kg, 0.15 mg/kg, 0.16 mg/kg, 0.17 mg/kg, 0.18 mg/kg, 0.19 mg/kg, or0.20 mg/kg.

In one embodiment, the dosage is between 0.005 mg/kg and 1.628 mg/kg.For example, the dsRNA can be administered at a dose of 0.0059 mg/kg,0.0295 mg/kg, 0.0590 mg/kg, 0.163 mg/kg, 0.543 mg/kg, 0.5900 mg/kg, or1.628 mg/kg.

In one embodiment, the dosage is between 0.2 mg/kg and 1.5 mg/kg. Forexample, the dsRNA can be administered at a dose of 0.2 mg/kg, 0.3mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg,1 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, or 1.5 mg/kg.

The dsRNA can be administered at a dose of 0.03 mg/kg, or 0.03, 0.1,0.2, or 0.4 mg/kg.

The pharmaceutical composition may be administered once daily or thedsRNA may be administered as two, three, or more sub-doses atappropriate intervals throughout the day or even using continuousinfusion or delivery through a controlled release formulation. In thatcase, the dsRNA contained in each sub-dose must be correspondinglysmaller in order to achieve the total daily dosage. The dosage unit canalso be compounded for delivery over several days, e.g., using aconventional sustained release formulation which provides sustainedrelease of the dsRNA over a several day period. Sustained releaseformulations are well known in the art and are particularly useful fordelivery of agents at a particular site, such as could be used with theagents of the present invention. In this embodiment, the dosage unitcontains a corresponding multiple of the daily dose.

The effect of a single dose on HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4,IL6R, BMP6, and/or NEO1 levels is long lasting, such that subsequentdoses are administered at not more than 3, 4, or 5 day intervals, or atnot more than 1, 2, 3, or 4 week intervals, or at not more than 5, 6, 7,8, 9, or 10 week intervals.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual dsRNAs encompassed by theinvention can be made using conventional methodologies or on the basisof in vivo testing using an appropriate animal model, as describedelsewhere herein.

Advances in mouse genetics have generated a number of mouse models forthe study of various human diseases, such as pathological processesmediated by HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/orNEO1 expression. Such models are used for in vivo testing of dsRNA, aswell as for determining a therapeutically effective dose. A suitablemouse model is, for example, a mouse containing a plasmid expressinghuman HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1.Another suitable mouse model is a transgenic mouse carrying a transgenethat expresses human HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6,and/or NEO1.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofcompositions featured in the invention lies generally within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the methods featured in the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range of the compound or, whenappropriate, of the polypeptide product of a target sequence (e.g.,achieving a decreased concentration of the polypeptide) that includesthe IC50 (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma may be measured, for example, by highperformance liquid chromatography.

The dsRNAs featured in the invention can be administered in combinationwith other known agents effective in treatment of pathological processesmediated by target gene expression. In any event, the administeringphysician can adjust the amount and timing of dsRNA administration onthe basis of results observed using standard measures of efficacy knownin the art or described herein.

Administration

The present invention also includes pharmaceutical compositions andformulations which include the dsRNA compounds featured in theinvention. The pharmaceutical compositions of the present invention maybe administered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical, pulmonary, e.g., by inhalation orinsufflation of powders or aerosols, including by nebulizer;intratracheal, intranasal, epidermal and transdermal, oral orparenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intraparenchymal, intrathecal orintraventricular, administration.

The dsRNA can be delivered in a manner to target a particular tissue.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Coated condoms, gloves and thelike may also be useful. Suitable topical formulations include those inwhich the dsRNAs featured in the invention are in admixture with atopical delivery agent such as lipids, liposomes, fatty acids, fattyacid esters, steroids, chelating agents and surfactants. Suitable lipidsand liposomes include neutral (e.g., dioleoylphosphatidyl DOPEethanolamine, dimyristoylphosphatidyl choline DMPC,distearoylphosphatidyl choline) negative (e.g., dimyristoylphosphatidylglycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAPand dioleoylphosphatidyl ethanolamine DOTMA). DsRNAs featured in theinvention may be encapsulated within liposomes or may form complexesthereto, in particular to cationic liposomes. Alternatively, dsRNAs maybe complexed to lipids, in particular to cationic lipids. Suitable fattyacids and esters include but are not limited to arachidonic acid, oleicacid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristicacid, palmitic acid, stearic acid, linoleic acid, linolenic acid,dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₁₀ alkyl ester (e.g., isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof. Topicalformulations are described in detail in U.S. Pat. No. 6,747,014, whichis incorporated herein by reference.

Liposomal Formulations

There are many organized surfactant structures besides microemulsionsthat have been studied and used for the formulation of drugs. Theseinclude monolayers, micelles, bilayers and vesicles. Vesicles, such asliposomes, have attracted great interest because of their specificityand the duration of action they offer from the standpoint of drugdelivery. As used in the present invention, the term “liposome” means avesicle composed of amphiphilic lipids arranged in a spherical bilayeror bilayers.

Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

Further advantages of liposomes include; liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomes start to merge with the cellular membranes and as themerging of the liposome and cell progresses, the liposomal contents areemptied into the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation asthe mode of delivery for many drugs. There is growing evidence that fortopical administration, liposomes present several advantages over otherformulations. Such advantages include reduced side-effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer a wide variety of drugs, both hydrophilic andhydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agentsincluding high-molecular weight DNA into the skin. Compounds includinganalgesics, antibodies, hormones and high-molecular weight DNAs havebeen administered to the skin. The majority of applications resulted inthe targeting of the upper epidermis

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged DNAmolecules to form a stable complex. The positively charged DNA/liposomecomplex binds to the negatively charged cell surface and is internalizedin an endosome. Due to the acidic pH within the endosome, the liposomesare ruptured, releasing their contents into the cell cytoplasm (Wang etal., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNArather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drugformulations to the skin. Application of liposomes containing interferonto guinea pig skin resulted in a reduction of skin herpes sores whiledelivery of interferon via other means (e.g., as a solution or as anemulsion) were ineffective (Weiner et al., Journal of Drug Targeting,1992, 2, 405-410). Further, an additional study tested the efficacy ofinterferon administered as part of a liposomal formulation to theadministration of interferon using an aqueous system, and concluded thatthe liposomal formulation was superior to aqueous administration (duPlessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G_(M1), or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al).

Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C_(1215G), thatcontains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al).U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948(Tagawa et al.) describe PEG-containing liposomes that can be furtherderivatized with functional moieties on their surfaces.

A number of liposomes comprising nucleic acids are known in the art. WO96/40062 to Thierry et al. discloses methods for encapsulating highmolecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 toTagawa et al. discloses protein-bonded liposomes and asserts that thecontents of such liposomes may include a dsRNA. U.S. Pat. No. 5,665,710to Rahman et al. describes certain methods of encapsulatingoligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. disclosesliposomes comprising dsRNAs targeted to the raf gene.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g., they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in Pharmaceutical Dosage Forms, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

Nucleic Acid Lipid Particles

In one embodiment, a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6,and/or NEO1 dsRNA featured in the invention is fully encapsulated in thelipid formulation, e.g., a nucleic acid lipid particle, e.g., SPLP,pSPLP, SNALP, or other nucleic acid-lipid particle. As used herein, theterm “SNALP” refers to a stable nucleic acid-lipid particle, includingSPLP. As used herein, the term “SPLP” refers to a nucleic acid-lipidparticle comprising plasmid DNA encapsulated within a lipid vesicle.SPLPs include “pSPLP,” which include an encapsulated condensingagent-nucleic acid complex as set forth in PCT Publication No. WO00/03683. Nucleic acid lipid particles typically contain a cationiclipid, a non-cationic lipid, and a lipid that prevents aggregation ofthe particle (e.g., a PEG-lipid conjugate). Nucleic acid lipid particlesare extremely useful for systemic applications, as they exhibit extendedcirculation lifetimes following intravenous (i.v.) injection andaccumulate at distal sites (e.g., sites physically separated from theadministration site).

The particles of the present invention typically have a mean diameter ofabout 50 nm to about 150 nm, more typically about 60 nm to about 130 nm,more typically about 70 nm to about 110 nm, most typically about 70 nmto about 90 nm, and are substantially nontoxic. In addition, the nucleicacids when present in the nucleic acid-lipid particles of the presentinvention are resistant in aqueous solution to degradation with anuclease. Nucleic acid-lipid particles and their method of preparationare disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484;6,586,410; 6,815,432; and PCT Publication No. WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g.,lipid to dsRNA ratio) will be in the range of from about 1:1 to about50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, fromabout 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 toabout 9:1. In some embodiments the lipid to dsRNA ratio can be about1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or 11:1.

In general, the lipid-nucleic acid particle is suspended in a buffer,e.g., PBS, for administration. In one embodiment, the pH of the lipidformulated siRNA is between 6.8 and 7.8, e.g., 7.3 or 7.4. Theosmolality can be, e.g., between 250 and 350 mOsm/kg, e.g., around 300,e.g., 298, 299, 300, 301, 302, 303, 304, or 305.

The cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammoniumchloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N—(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),N—(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-Dioleylamino)-1,2-propanedio (DOAP),1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA),2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) oranalogs thereof,(3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine(ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (MC3),1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol(C12-200 or Tech G1), or a mixture thereof. The cationic lipid maycomprise from about 20 mol % to about 50 mol % or about 40 mol % of thetotal lipid present in the particle.

The non-cationic lipid may be an anionic lipid or a neutral lipidincluding, but not limited to, distearoylphosphatidylcholine (DSPC),dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine(DPPC), dioleoylphosphatidylglycerol (DOPG),dipalmitoylphosphatidylglycerol (DPPG),dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoylphosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or amixture thereof. The non-cationic lipid may be from about 5 mol % toabout 90 mol %, about 10 mol %, or about 58 mol % if cholesterol isincluded, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles may be, forexample, a polyethyleneglycol (PEG)-lipid including, without limitation,a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), aPEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. ThePEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (Ci₂), aPEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (C1₆), or aPEG-distearyloxypropyl (C₁₈). Other examples of PEG conjugates includePEG-cDMA (N-[(methoxy poly(ethyleneglycol)2000)carbamyl]-1,2-dimyristyloxlpropyl-3-amine), mPEG2000-DMG(mPEG-dimyrystylglycerol (with an average molecular weight of 2,000) andPEG-C-DOMG (R-3-[(ω-methoxy-poly(ethyleneglycol)2000)carbamoyl)]-1,2-dimyristyloxlpropyl-3-amine). The conjugatedlipid that prevents aggregation of particles may be from 0 mol % toabout 20 mol % or about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9, or 2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includescholesterol at, e.g., about 10 mol % to about 60 mol % or about 45, 46,47, 48, 49, 50, 51, 52, 53, 54, or 55 mol % of the total lipid presentin the particle.

LNP01

LNP01 formulations are described, e.g., in International ApplicationPublication No. WO 2008/042973, which is hereby incorporated byreference.

Additional exemplary formulations are described in Table A.

TABLE A cationic lipid/non-cationic lipid/cholesterol/PEG-lipidconjugate Cationic Mol % ratios Lipid Lipid:siRNA ratio SNALP DLinDMADLinDMA/DPPC/Cholesterol/PEG-cDMA (57.1/7.1/34.4/1.4) lipid:siRNA ~7:1S-XTC XTC XTC/DPPC/Cholesterol/PEG-cDMA 57.1/7.1/34.4/1.4 lipid:siRNA~7:1 LNP05 XTC XTC/DSPC/Cholesterol/PEG-DMG 57.5/7.5/31.5/3.5lipid:siRNA ~6:1 LNP06 XTC XTC/DSPC/Cholesterol/PEG-DMG57.5/7.5/31.5/3.5 lipid:siRNA ~11:1 LNP07 XTCXTC/DSPC/Cholesterol/PEG-DMG 60/7.5/31/1.5, lipid:siRNA ~6:1 LNP08 XTCXTC/DSPC/Cholesterol/PEG-DMG 60/7.5/31/1.5, lipid:siRNA ~11:1 LNP09 XTCXTC/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA 10:1 LNP10ALN100 ALN100/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA 10:1LNP11 MC3 MC-3/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA 10:1LNP12 C12-200 C12-200/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5Lipid:siRNA 10:1 LNP13 XTC XTC/DSPC/Chol/PEG-DMG 50/10/38.5/1.5Lipid:siRNA: 33:1 LNP14 MC3 MC3/DSPC/Chol/PEG-DMG 40/15/40/5Lipid:siRNA: 11:1 LNP15 MC3 MC3/DSPC/Chol/PEG-DSG/GalNAc-PEG-DSG50/10/35/4.5/0.5 Lipid:siRNA: 11:1 LNP16 MC3 MC3/DSPC/Chol/PEG-DMG50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP17 MC3 MC3/DSPC/Chol/PEG-DSG50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP18 MC3 MC3/DSPC/Chol/PEG-DMG50/10/38.5/1.5 Lipid:siRNA: 12:1 LNP19 MC3 MC3/DSPC/Chol/PEG-DMG50/10/35/5 Lipid:siRNA: 8:1 LNP20 MC3 MC3/DSPC/Chol/PEG-DPG50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP21 C12-200 C12-200/DSPC/Chol/PEG-DSG50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP22 XTC XTC/DSPC/Chol/PEG-DSG50/10/38.5/1.5 Lipid:siRNA: 10:1

SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprisingformulations are described in International Publication No.WO2009/127060, filed Apr. 15, 2009, which is hereby incorporated byreference.

XTC (2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane) comprisingformulations are described, e.g., in U.S. Provisional Ser. No.61/148,366, filed Jan. 29, 2009; U.S. Provisional Ser. No. 61/156,851,filed Mar. 2, 2009; U.S. Provisional Serial No. filed Jun. 10, 2009;U.S. Provisional Ser. No. 61/228,373, filed Jul. 24, 2009; U.S.Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, and InternationalApplication No. PCT/US2010/022614, filed Jan. 29, 2010, which are herebyincorporated by reference.

MC3 ((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate), (e.g., DLin-M-C3-DMA) comprisingformulations are described, e.g., in U.S. Provisional Ser. No.61/244,834, filed Sep. 22, 2009, U.S. Provisional Ser. No. 61/185,800,filed Jun. 10, 2009, and International Application No. PCT/US10/28224,filed Jun. 10, 2010, which are hereby incorporated by reference.

ALNY-100((3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine)comprising formulations are described, e.g., International patentapplication number PCT/US09/63933, filed on Nov. 10, 2009, which ishereby incorporated by reference.

C12-200, i.e., Tech G1, comprising formulations are described in U.S.Provisional Ser. No. 61/175,770, filed May 5, 2009 and InternationalApplication No. PCT/US10/33777, filed May 5, 2010, which are herebyincorporated by reference.

Formulations prepared by either the standard or extrusion-free methodcan be characterized in similar manners. For example, formulations aretypically characterized by visual inspection. They should be whitishtranslucent solutions free from aggregates or sediment. Particle sizeand particle size distribution of lipid-nanoparticles can be measured bylight scattering using, for example, a Malvern Zetasizer Nano ZS(Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nmin size. The particle size distribution should be unimodal. The totalsiRNA concentration in the formulation, as well as the entrappedfraction, is estimated using a dye exclusion assay. A sample of theformulated siRNA can be incubated with an RNA-binding dye, such asRibogreen (Molecular Probes) in the presence or absence of a formulationdisrupting surfactant, e.g., 0.5% Triton-X100. The total siRNA in theformulation can be determined by the signal from the sample containingthe surfactant, relative to a standard curve. The entrapped fraction isdetermined by subtracting the “free” siRNA content (as measured by thesignal in the absence of surfactant) from the total siRNA content.Percent entrapped siRNA is typically >85%. For a nucleic acid lipidformulation, the particle size is at least 30 nm, at least 40 nm, atleast 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90nm, at least 100 nm, at least 110 nm, and at least 120 nm. The suitablerange is typically about at least 50 nm to about at least 110 nm, aboutat least 60 nm to about at least 100 nm, or about at least 80 nm toabout at least 90 nm.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. In some embodiments, oralformulations are those in which dsRNAs featured in the invention areadministered in conjunction with one or more penetration enhancerssurfactants and chelators. Suitable surfactants include fatty acidsand/or esters or salts thereof, bile acids and/or salts thereof.Suitable bile acids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitablefatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g., sodium). In some embodiments, combinations of penetrationenhancers are used, for example, fatty acids/salts in combination withbile acids/salts. One exemplary combination is the sodium salt of lauricacid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAsfeatured in the invention may be delivered orally, in granular formincluding sprayed dried particles, or complexed to form micro ornanoparticles. DsRNA complexing agents include poly-amino acids;polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Suitable complexing agents include chitosan,N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine,polyspermines, protamine, polyvinylpyridine,polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g.,p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor dsRNAs and their preparation are described in detail in U.S. Pat.No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014,each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into thebrain), intrathecal, intraventricular or intrahepatic administration mayinclude sterile aqueous solutions which may also contain buffers,diluents and other suitable additives such as, but not limited to,penetration enhancers, carrier compounds and other pharmaceuticallyacceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids. Particularlypreferred are formulations that target the liver when treating hepaticdisorders such as hepatic carcinoma.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Emulsions

The compositions of the present invention may be prepared and formulatedas emulsions. Emulsions are typically heterogeneous systems of oneliquid dispersed in another in the form of droplets usually exceeding0.1 μm in diameter (Idson, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 2, p. 335; Higuchi et al., in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions areoften biphasic systems comprising two immiscible liquid phasesintimately mixed and dispersed with each other. In general, emulsionsmay be of either the water-in-oil (w/o) or the oil-in-water (o/w)variety. When an aqueous phase is finely divided into and dispersed asminute droplets into a bulk oily phase, the resulting composition iscalled a water-in-oil (w/o) emulsion. Alternatively, when an oily phaseis finely divided into and dispersed as minute droplets into a bulkaqueous phase, the resulting composition is called an oil-in-water (o/w)emulsion. Emulsions may contain additional components in addition to thedispersed phases, and the active drug which may be present as a solutionin either the aqueous phase, oily phase or itself as a separate phase.Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, andanti-oxidants may also be present in emulsions as needed. Pharmaceuticalemulsions may also be multiple emulsions that are comprised of more thantwo phases such as, for example, in the case of oil-in-water-in-oil(o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complexformulations often provide certain advantages that simple binaryemulsions do not. Multiple emulsions in which individual oil droplets ofan o/w emulsion enclose small water droplets constitute a w/o/wemulsion. Likewise a system of oil droplets enclosed in globules ofwater stabilized in an oily continuous phase provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York,N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group: nonionic, anionic, cationic andamphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 199). Emulsion formulations for oral delivery have beenvery widely used because of ease of formulation, as well as efficacyfrom an absorption and bioavailability standpoint (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil baselaxatives, oil-soluble vitamins and high fat nutritive preparations areamong the materials that have commonly been administered orally as o/wemulsions.

In one embodiment of the present invention, the compositions of dsRNAsand nucleic acids are formulated as microemulsions. A microemulsion maybe defined as a system of water, oil and amphiphile which is a singleoptically isotropic and thermodynamically stable liquid solution(Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245).Typically microemulsions are systems that are prepared by firstdispersing an oil in an aqueous surfactant solution and then adding asufficient amount of a fourth component, generally an intermediatechain-length alcohol to form a transparent system. Therefore,microemulsions have also been described as thermodynamically stable,isotropically clear dispersions of two immiscible liquids that arestabilized by interfacial films of surface-active molecules (Leung andShah, in: Controlled Release of Drugs: Polymers and Aggregate Systems,Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215).Microemulsions commonly are prepared via a combination of three to fivecomponents that include oil, water, surfactant, cosurfactant andelectrolyte. Whether the microemulsion is of the water-in-oil (w/o) oran oil-in-water (o/w) type is dependent on the properties of the oil andsurfactant used and on the structure and geometric packing of the polarheads and hydrocarbon tails of the surfactant molecules (Schott, inRemington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.,1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared toconventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Sci., 1996, 85, 138-143). Often microemulsions may form spontaneouslywhen their components are brought together at ambient temperature. Thismay be particularly advantageous when formulating thermolabile drugs,peptides or dsRNAs. Microemulsions have also been effective in thetransdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of dsRNAs and nucleic acids from thegastrointestinal tract, as well as improve the local cellular uptake ofdsRNAs and nucleic acids.

Microemulsions of the present invention may also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the dsRNAs and nucleicacids of the present invention. Penetration enhancers used in themicroemulsions of the present invention may be classified as belongingto one of five broad categories—surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly dsRNAs, to the skin of animals. Most drugs are present insolution in both ionized and nonionized forms. However, usually onlylipid soluble or lipophilic drugs readily cross cell membranes. It hasbeen discovered that even non-lipophilic drugs may cross cell membranesif the membrane to be crossed is treated with a penetration enhancer. Inaddition to aiding the diffusion of non-lipophilic drugs across cellmembranes, penetration enhancers also enhance the permeability oflipophilic drugs.

Penetration enhancers may be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (Lee et al., Critical Reviewsin Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the abovementioned classes of penetration enhancers are described below ingreater detail.

Surfactants: In connection with the present invention, surfactants (or“surface-active agents”) are chemical entities which, when dissolved inan aqueous solution, reduce the surface tension of the solution or theinterfacial tension between the aqueous solution and another liquid,with the result that absorption of dsRNAs through the mucosa isenhanced. In addition to bile salts and fatty acids, these penetrationenhancers include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al.,J. Pharm. Pharmacol., 1988, 40, 252).

Fatty acids: Various fatty acids and their derivatives which act aspenetration enhancers include, for example, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C.sub.1-10 alkyl esters thereof (e.g., methyl, isopropyland t-butyl), and mono- and di-glycerides thereof (i.e., oleate,laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems,1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44,651-654).

Bile salts: The physiological role of bile includes the facilitation ofdispersion and absorption of lipids and fat-soluble vitamins (Brunton,Chapter 38 in: Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996,pp. 934-935). Various natural bile salts, and their syntheticderivatives, act as penetration enhancers. Thus the term “bile salts”includes any of the naturally occurring components of bile as well asany of their synthetic derivatives. Suitable bile salts include, forexample, cholic acid (or its pharmaceutically acceptable sodium salt,sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholicacid (sodium deoxycholate), glucholic acid (sodium glucholate),glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodiumglycodeoxycholate), taurocholic acid (sodium taurocholate),taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid(sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodiumtauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate andpolyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto etal., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm.Sci., 1990, 79, 579-583).

Chelating Agents: Chelating agents, as used in connection with thepresent invention, can be defined as compounds that remove metallic ionsfrom solution by forming complexes therewith, with the result thatabsorption of dsRNAs through the mucosa is enhanced. With regards totheir use as penetration enhancers in the present invention, chelatingagents have the added advantage of also serving as DNase inhibitors, asmost characterized DNA nucleases require a divalent metal ion forcatalysis and are thus inhibited by chelating agents (Jarrett, J.Chromatogr., 1993, 618, 315-339). Suitable chelating agents include butare not limited to disodium ethylenediaminetetraacetate (EDTA), citricacid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate andhomovanilate), N-acyl derivatives of collagen, laureth-9 and N-aminoacyl derivatives of beta-diketones (enamines)(Lee et al., CriticalReviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi,Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33;Buur et al., J. Control Rel., 1990, 14, 43-51).

Non-chelating non-surfactants: As used herein, non-chelatingnon-surfactant penetration enhancing compounds can be defined ascompounds that demonstrate insignificant activity as chelating agents oras surfactants but that nonetheless enhance absorption of dsRNAs throughthe alimentary mucosa (Muranishi, Critical Reviews in Therapeutic DrugCarrier Systems, 1990, 7, 1-33). This class of penetration enhancersinclude, for example, unsaturated cyclic ureas, 1-alkyl- and1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, page 92); and non-steroidalanti-inflammatory agents such as diclofenac sodium, indomethacin andphenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39,621-626).

Carriers

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate dsRNA in hepatic tissue can be reduced when it iscoadministered with polyinosinic acid, dextran sulfate, polycytidic acidor 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao etal., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl.Acid Drug Dev., 1996, 6, 177-183.

Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids may includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

Other Components

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions may contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or may contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions may contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in theinvention include (a) one or more dsRNA compounds and (b) one or moreanti-cytokine biologic agents which function by a non-RNAi mechanism.Examples of such biologics include, biologics that target IL1β (e.g.,anakinra), IL6 (tocilizumab), or TNF (etanercept, infliximab, adlimumab,or certolizumab).

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofcompositions featured in the invention lies generally within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the methods featured in the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range of the compound or, whenappropriate, of the polypeptide product of a target sequence (e.g.,achieving a decreased concentration of the polypeptide) that includesthe IC50 (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma may be measured, for example, by highperformance liquid chromatography.

In addition to their administration, as discussed above, the dsRNAsfeatured in the invention can be administered in combination with otherknown agents effective in treatment of pathological processes mediatedby HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1expression. In any event, the administering physician can adjust theamount and timing of dsRNA administration on the basis of resultsobserved using standard measures of efficacy known in the art ordescribed herein.

Methods for Inhibiting Expression of a HAMP, HFE2, HFE, TFR2, BMPR1a,SMAD4, IL6R, BMP6, and/or NEO1 Gene

In yet another aspect, the invention provides a method for inhibitingthe expression of a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6,and/or NEO1 gene in a mammal. The method includes administering acomposition featured in the invention to the mammal such that expressionof the target HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/orNEO1 gene is silenced.

When the organism to be treated is a mammal such as a human, thecomposition may be administered by any means known in the art including,but not limited to oral or parenteral routes, including intracranial(e.g., intraventricular, intraparenchymal and intrathecal), intravenous,intramuscular, subcutaneous, transdermal, airway (aerosol), nasal,rectal, and topical (including buccal and sublingual) administration. Incertain embodiments, the compositions are administered by intravenousinfusion or injection.

Methods for Treating Diseases Caused by Expression of a HAMP, HFE2, HFE,TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 Gene

The invention relates in particular to the use of a dsRNA targetingHAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 andcompositions containing at least one such dsRNA for the treatment of aHAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1-mediateddisorder or disease. For example, the compositions described herein canbe used to treat anemia and other diseases associated with lowered ironlevels.

Methods of Using dsRNAs Targeting HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4,IL6R, BMP6, and/or NEO1

In one aspect, the invention provides use of a siRNA for inhibiting theexpression of HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/orNEO1 in a mammal. The method includes administering a composition of theinvention to the mammal such that expression of the target HAMP, HFE2,HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene is decreased. Insome embodiments, HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6,and/or NEO1 expression is decreased for an extended duration, e.g., atleast one week, two weeks, three weeks, or four weeks or longer. Forexample, in certain instances, expression of the HAMP, HFE2, HFE, TFR2,BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene is suppressed by at leastabout 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% byadministration of a siRNA described herein. In some embodiments, theHAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene issuppressed by at least about 60%, 70%, or 80% by administration of thesiRNA. In some embodiments, the HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4,IL6R, BMP6, and/or NEO1 gene is suppressed by at least about 85%, 90%,or 95% by administration of the double-stranded oligonucleotide.

The methods and compositions described herein can be used to treatdiseases and conditions that can be modulated by down regulating HAMP,HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene expression.For example, the compositions described herein can be used to treatanemia and other forms of iron imbalance such as refractory anemia,refractory anemia of chronic disease (ACD), iron-restrictederythropoiesis, and the pathological conditions associated with thesedisorders. In some aspects, ACD subjects are those who are refractory toESAs and i.v. iron administration. In some embodiments, the methodincludes administering an effective amount of a siRNA disclosed hereinto a patient having lower iron levels relative to a control patient.

Therefore, the invention also relates to the use of a siRNA for thetreatment of a disorder or disease mediated by or related to HAMP, HFE2,HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene expression. Forexample, a siRNA is used for treatment of anemia.

The effect of the decreased HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R,BMP6, and/or NEO1 gene expression preferably results in an enhancementof iron mobilization in the mammal. In some embodiments, ironmobilization is enhanced by at least 10%, 15%, 20%, 25%, 30%, 40%, 50%,or 60%, or more, as compared to pretreatment levels.

The effect of the decreased HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R,BMP6, and/or NEO1 gene expression preferably results in an Hb increasein the mammal. In some embodiments, Hb is increased by at least 10%,15%, 20%, 25%, 30%, 40%, 50%, or 60%, or more, as compared topretreatment levels.

The effect of the decreased HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R,BMP6, and/or NEO1 gene expression preferably results in a serum ironincrease in the mammal. In some embodiments, serum iron is increased byat least 10%, 15%, 20%, 25%, 30%, 40%, 50%, or 60%, or more, as comparedto pretreatment levels.

The effect of the decreased HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R,BMP6, and/or NEO1 gene expression preferably results in a transderrin(Tf) saturation increase in the mammal. In some embodiments, Tfsaturation is increased by at least 10%, 15%, 20%, 25%, 30%, 40%, 50%,or 60%, or more, as compared to pretreatment levels.

The effect of the decreased HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R,BMP6, and/or NEO1 gene expression preferably results in decreased levelsof HAMP in the mammal. In some embodiments, HAMP is decreased by atleast 10%, 15%, 20%, 25%, 30%, 40%, 50%, or 60%, or more, as compared topretreatment levels.

The method includes administering a siRNA to the subject to be treated.The subject to be treated is generally a subject in need thereof. Whenthe subject to be treated is a mammal, such as a human, the compositioncan be administered by any means known in the art including, but notlimited to oral or parenteral routes, including intravenous,intramuscular, subcutaneous, transdermal, and airway (aerosol)administration. In some embodiments, the compositions are administeredby intravenous infusion or injection.

The method includes administering a siRNA, e.g., a dose sufficient todepress levels of HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6,and/or NEO1 mRNA for at least 5, more preferably 7, 10, 14, 21, 25, 30or 40 days; and optionally, administering a second single dose of dsRNA,wherein the second single dose is administered at least 5, morepreferably 7, 10, 14, 21, 25, 30 or 40 days after the first single doseis administered, thereby inhibiting the expression of the target gene ina subject.

In one embodiment, doses of siRNA are administered not more than onceevery four weeks, not more than once every three weeks, not more thanonce every two weeks, or not more than once every week. In anotherembodiment, the administrations can be maintained for one, two, three,or six months, or one year or longer.

In another embodiment, administration can be provided when Hb levelsreach or drop lower than a predetermined minimal level, such as lessthan 8 g/dL, 9 g/dL, or 10 g/dL. In some aspects, administration iscontinued until Hb levels are >11 g/dL, e.g, 12 g/dL.

In another embodiment, administration can be provided when a patientpresents with various known symptoms of disorders such as anemia. Thesecan include fatigue, shortness of breath, headache, dizziness, or paleskin.

In another embodiment, administration can be provided when a patient isdiagnosed with anemia via CBC.

In general, the siRNA does not activate the immune system, e.g., it doesnot increase cytokine levels, such as TNF-alpha or IFN-alpha levels. Forexample, when measured by an assay, such as an in vitro PBMC assay, suchas described herein, the increase in levels of TNF-alpha or IFN-alpha,is less than 30%, 20%, or 10% of control cells treated with a controldsRNA, such as a dsRNA that does not target HAMP, HFE2, HFE, TFR2,BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1.

In an aspect, a subject can be administered a therapeutic amount ofsiRNA, such as 0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2.0 mg/kg, or 2.5 mg/kgdsRNA. The siRNA can be administered by intravenous infusion over aperiod of time, such as over a 5 minute, 10 minute, 15 minute, 20minute, or 25 minute period. The administration is repeated, forexample, on a regular basis, such as biweekly (i.e., every two weeks)for one month, two months, three months, four months or longer. After aninitial treatment regimen, the treatments can be administered on a lessfrequent basis. For example, after administration biweekly for threemonths, administration can be repeated once per month, for six months ora year or longer. Administration of the siRNA can reduce HAMP, HFE2,HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 levels, e.g., in acell, tissue, blood, urine or other compartment of the patient by atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80% or atleast 90% or more.

Before administration of a full dose of the siRNA, patients can beadministered a smaller dose, such as a 5% infusion reaction, andmonitored for adverse effects, such as an allergic reaction, or forelevated lipid levels or blood pressure. In another example, the patientcan be monitored for unwanted immunostimulatory effects, such asincreased cytokine (e.g., TNF-alpha or IFN-alpha) levels.

A treatment or preventive effect is evident when there is astatistically significant improvement in one or more parameters ofdisease status, or by a failure to worsen or to develop symptoms wherethey would otherwise be anticipated. As an example, a favorable changeof at least 10% in a measurable parameter of disease, and preferably atleast 20%, 30%, 40%, 50% or more can be indicative of effectivetreatment. Efficacy for a given siRNA drug or formulation of that drugcan also be judged using an experimental animal model for the givendisease as known in the art. When using an experimental animal model,efficacy of treatment is evidenced when a statistically significantreduction in a marker or symptom is observed.

Additional Agents and Co-Administration

In further embodiments, administration of a siRNA is administered incombination an additional therapeutic agent. The siRNA and an additionaltherapeutic agent can be administered in combination in the samecomposition, e.g., parenterally, or the additional therapeutic agent canbe administered as part of a separate composition or by another methoddescribed herein.

In one embodiment, the siRNA is administered to the patient, and thenthe additional therapeutic agent is administered to the patient (or viceversa). In another embodiment, the siRNA and the additional therapeuticagent are administered at the same time.

In some aspects, the additional agent can include one or moreErythropoiesis-stimulating agents (ESAs). ESAs are generally known inthe art. ESAs can include Erythropoietin (EPO), Epoetin alfa(Procrit/Epogen), Epoetin beta (NeoRecormon), Darbepoetin alfa(Aranesp), and Methoxy polyethylene glycol-epoetin beta (Micera). ESAscan be administered in various doses, e.g., 7,000 U/week to 30,000U/week.

In some aspects, the additional agent can include intravenous iron. Ironcan be administered in various doses known in the art.

In some aspects, two or more dsRNAs are co-administered to a subject. Inone embodiment, a first dsRNA is administered to the patient, and then asecond dsRNA is administered to the patient (or vice versa). In anotherembodiment, the first dsRNA and the second dsRNA are administered at thesame time.

In some aspects, a HAMP dsRNA is co-administered with one or more dsRNAsselected from HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/orNEO1dsRNAs.

In some aspects, a HFE2 dsRNA is co-administered with one or more dsRNAsselected from HAMP, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/orNEO1dsRNAs.

In some aspects, a HFE dsRNA is co-administered with one or more dsRNAsselected from HAMP, HFE2, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/orNEO1dsRNAs.

In some aspects, a TFR2 dsRNA is co-administered with one or more dsRNAsselected from HAMP, HFE2, HFE, BMPR1a, SMAD4, IL6R, BMP6, and/orNEO1dsRNAs.

In some aspects, a BMPR1a dsRNA is co-administered with one or moredsRNAs selected from HAMP, HFE2, HFE, TFR2, SMAD4, IL6R, BMP6, and/orNEO1dsRNAs.

In some aspects, a SMAD4 dsRNA is co-administered with one or moredsRNAs selected from HAMP, HFE2, HFE, TFR2, BMPR1a, IL6R, BMP6, and/orNEO1dsRNAs.

In some aspects, an IL6R dsRNA is co-administered with one or moredsRNAs selected from HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, BMP6, and/orNEO1dsRNAs.

In some aspects, a BMP6 dsRNA is co-administered with one or more dsRNAsselected from HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, and/orNEO1dsRNAs.

In some aspects, a NEO1 dsRNA is co-administered with one or more dsRNAsselected from HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, and/or BMP6dsRNAs.

In another aspect, the invention features, a method of instructing anend user, e.g., a caregiver or a subject, on how to administer a siRNAdescribed herein. The method includes, optionally, providing the enduser with one or more doses of the siRNA, and instructing the end userto administer the siRNA on a regimen described herein, therebyinstructing the end user.

Identification of Subjects in Need of dsRNA Administration

In one aspect, the invention provides a method of treating a patient byselecting a patient on the basis that the patient is in need of ironmobilization. The method includes administering to the patient a siRNAin an amount sufficient to increase the patient's iron mobilization.

In one aspect, the invention provides a method of treating a patient byselecting a patient on the basis that the patient is in need ofincreased Hb levels. Such a subject can have Hb levels of <9 g/dL. Themethod includes administering to the patient a siRNA in an amountsufficient to increase the patient's Hb levels. Typically target Hblevels are >11 g/dL, e.g., 11 g/dL or 12 g/dL.

In some aspects, a subject is identified as having anemia. In someaspects, a subject is identified as having a refractory form of anemia.In some aspects, a subject is identified as having ACD. Such subjectscan be in need of administration of a dsRNA described herein. ACD caninclude a form of anemia wherein the subject is refractory to ESAsand/or i.v. iron administration. Typical clinical presentation of ACDincludes fatigue, shortness of breadth, headache, dizziness, and/or paleskin. ACD can also be diagnosed via a CBC test, which is generally knownin the art. ACD can also be diagnosed via serum iron levels, Tfsaturation, and/or ferritin levels. ACD is typically diagnosed incertain settings such as subjects with CKD, cancer, chronic inflammatorydiseases such as RA, or IRIDA. In some aspects, a subject with ACD hasHb levels of less than 9 g/dL. Such subjects typically becomesymptomatic for ACD.

CKD can result in reduced renal EPO synthesis, dietary hematinicdeficiencies, blood loss, and/or elevated hepcidin levels. The elevationin hepcidin levels can be due to decreased renal excretion and/or lowgrade inflammation characterized by, e.g., interleukin (IL)-6.

In some aspects, a subject is identified as having iron-restrictederythropoiesis (IRE). Such subjects can be in need of administration ofa dsRNA described herein. IRE can be assessed via reticulocyte Hb (CHr).Typically a result of <28 pg suggests IRE, where normal is in the rangeof 28-35 pg. IRE can also be assessed via percent (%) hypochromic RBCs.Typically a result of >10% suggests IRE, where 1-5% is generallyconsidered normal.

A healthcare provider, such as a doctor, nurse, or family member, cantake a family history before prescribing or administering a siRNA. Inaddition, a test may be performed to determine a geneotype or phenotype.For example, a DNA test may be performed on a sample from the patient,e.g., a blood sample, to identify the relevant genotype and/or phenotypebefore a dsRNA is administered to the patient. In another embodiment, atest is performed to identify a related genotype and/or phenotype.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the dsRNAs and methods featured in the invention,suitable methods and materials are described below. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and notintended to be limiting.

EXAMPLES Example 1 dsRNA Synthesis Source of Reagents

Where the source of a reagent is not specifically given herein, suchreagent may be obtained from any supplier of reagents for molecularbiology at a quality/purity standard for application in molecularbiology.

siRNA Synthesis

Single-stranded RNAs were produced by solid phase synthesis on a scaleof 1 μmole using an Expedite 8909 synthesizer (Applied Biosystems,Applera Deutschland GmbH, Darmstadt, Germany) and controlled pore glass(CPG, 500 Å, Proligo Biochemie GmbH, Hamburg, Germany) as solid support.RNA and RNA containing 2′-O-methyl nucleotides were generated by solidphase synthesis employing the corresponding phosphoramidites and2′-O-methyl phosphoramidites, respectively (Proligo Biochemie GmbH,Hamburg, Germany). These building blocks were incorporated at selectedsites within the sequence of the oligoribonucleotide chain usingstandard nucleoside phosphoramidite chemistry such as described inCurrent protocols in nucleic acid chemistry, Beaucage, S. L. et al.(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA. Phosphorothioatelinkages were introduced by replacement of the iodine oxidizer solutionwith a solution of the Beaucage reagent (Chruachem Ltd, Glasgow, UK) inacetonitrile (1%). Further ancillary reagents were obtained fromMallinckrodt Baker (Griesheim, Germany).

Deprotection and purification of the crude oligoribonucleotides by anionexchange HPLC were carried out according to established procedures.Yields and concentrations were determined by UV absorption of a solutionof the respective RNA at a wavelength of 260 nm using a spectralphotometer (DU 640B, Beckman Coulter GmbH, Unterschleiβheim, Germany).Double stranded RNA was generated by mixing an equimolar solution ofcomplementary strands in annealing buffer (20 mM sodium phosphate, pH6.8; 100 mM sodium chloride), heated in a water bath at 85-90° C. for 3minutes and cooled to room temperature over a period of 3-4 hours. Theannealed RNA solution was stored at −20° C. until use.

Nucleic acid sequences are represented below using standardnomenclature, and specifically the abbreviations of Table 1.

TABLE 1 Abbreviations Abbreviation Nucleotide(s) Aadenosine-3′-phosphate C cytidine-3′-phosphate G guanosine-3′-phosphateU uridine-3′-phosphate N any nucleotide (G, A, C, or T) a2′-O-methyladenosine-3′-phosphate c 2′-O-methylcytidine-3′-phosphate g2′-O-methylguanosine-3′-phosphate u 2′-O-methyluridine-3′-phosphate T,dT 2′-deoxythymidine-3′-phosphate sT; sdT2′-deoxy-thymidine-5′phosphate-phosphorothioate Af2′-fluoroadenosine-3′-phosphate Cf 2′-fluorocytidine-3′-phosphate Gf2′-fluoroguanosine-3′-phosphate Uf 2′-fluorouridine-3′-phosphate

Example 2 HAMP siRNA Design Transcripts

siRNA design was carried out to identify siRNAs targeting human,cynomolgus monkey (Macaca fascicularis; herein “cyno”), mouse, and ratHAMP transcripts annotated in the NCBI Gene databasencbi.nlm.nih.gov/gene website. In mouse, the HAMP gene is duplicated,yielding distinct HAMP1 and HAMP2 loci; duplex designs targeted onlyHAMP1. Design used the following transcripts from the NCBI RefSeq andGenBank collections: Human—NM_021175.2 (SEQ ID NO:1); Cyno—EU076443.1;Mouse—NM_032541.1; Rat—NM_053469.1. Due to the short length of the HAMPtranscripts and the high degree of primate/rodent HAMP sequencedivergence, siRNA duplexes were designed in multiple separate batches.The separate batches are listed below and matched the various species asfollows:

-   -   human and cyno HAMP, exactly;    -   only human HAMP, exactly;    -   human and cyno HAMP, with mismatches to HAMP in both species        allowed at sense-strand position 19 when a G or C HAMP        targeting-nucleotide was replaced with a U or A, i.e. “UA-swap”;    -   human and cyno HAMP, with exact match to human HAMP and        mismatches to cyno HAMP allowed at sense-strand positions 1, 2,        and 19, i.e. “mismatch-to-cyno”;    -   mouse HAMP1, exactly;    -   only rat HAMP, exactly.

All siRNA duplexes were designed that shared 100% identity with alllisted human, cyno, mouse, or rat transcripts with the exception(s) ofdesignated mismatched-to-target bases. Unless otherwise noted, duplexesthemselves were 100% complementary and double-stranded.

siRNA Design, Specificity, and Efficacy Prediction

The predicted specificity of all possible 19mers was predicted from eachsequence. Candidate 19mers were selected that lacked repeats longer than7 nucleotides. These siRNAs were used in comprehensive searches againstthe appropriate transcriptomes.

siRNAs strands were assigned to a category of specificity according tothe calculated scores: a score above 3 qualifies as highly specific,equal to 3 as specific and between 2.2 and 2.8 as moderately specific.We sorted by the specificity of the antisense strand.

Table 2 provides the sequences of the sense and antisense strands of 42duplexes targeting the 3′UTR of the human HAMP gene.

Table 3 provides the sequences of the sense and antisense strands of 47duplexes targeting the CDS of the human HAMP gene.

Table 4 provides the sequences of the sense and antisense strands of themodified duplexes targeting the HAMP gene.

Table 5 provides the sequences of the sense and antisense strands of theunmodified version of the duplexes shown in Table 4.

The antisense-derived human/cyno, mouse, rat, UA-swap, andmismatch-to-cyno oligonucleotides shown in Tables 3-4 were synthesizedand formed into duplexes.

In some instances the duplexes contained no chemical modifications(unmodified).

In some instances the duplexes contained modifications (modified). Forexample, some duplexes were made with “Light Fluoro” chemicalmodifications as follows: all pyrimidines (cytosine and uridine) in thesense strand were replaced with corresponding 2′-Fluoro bases (2′ FluoroC and 2′-Fluoro U). In the antisense strand, pyrimidines adjacent to(towards 5′ position) ribo A nucleoside was replaced with theircorresponding 2-Fluoro nucleosides.

Example 3 HAMP siRNA Screening Cell Culture and Transfections: DualLuciferase System:

COS7 cells (ATCC, Manassas, Va.) were grown to near confluence at 37° C.in an atmosphere of 5% CO₂ in DMEM (Gibco) supplemented with 10% FBSbefore being released from the plate by trypsinization. Cells weretransfected with a psiCHECK2 vector (Promega) containing the human HAMPopen reading frame (ORF). The ORF was introduced following the stopcodon in the renilla luciferase sequence. Plasmid transfection wascarried out by adding 19.8 μl of Opti-MEM plus 0.2 μl of LipofectamineRNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) and 2.5 ngplasmid into a 96-well plate and incubated at room temperature for 15minutes. 80 μl of complete growth media containing ˜2×10⁴ COS7 cellswere then added. Cells were incubated for three hours, after which themedia was removed from the wells and replaced with 80 μl of completegrowth media. Transfection of siRNA was accomplished out by preparingadding 14.8 μl of Opti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well(Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl of siRNA duplexesper well into a new 96-well plate and incubated at room temperature for15 minutes. The 20 μl volumes containing the lipoplexes were then addedover the culture plates and incubated for 48 hours. Single doseexperiments were performed at final concentrations of 10 nM and 0.1 nM.An additional concentration of 0.01 nM was performed for selectedduplexes. Final duplex concentrations for dose response experiments were10, 1.67, 0.278, 0.046, 0.0077, 0.0012, 0.0002, and 0.000035 nM.

Endogenous System (Human):

For HAMP, HepG2 cells were used. HepG2 cells (ATCC, Manassas, Va.) weregrown to near confluence at 37° C. in an atmosphere of 5% CO₂ in MEM(Gibco) supplemented with 10% FBS before being released from the plateby trypsinization. Transfection was carried out by adding 14.8 μl ofOpti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen,Carlsbad Calif. cat #13778-150) to 5 μl of siRNA duplexes per well intoa 96-well plate and incubated at room temperature for 15 minutes. 80 μlof complete growth media without antibiotic containing ˜2×10⁴ HepG2cells were then added to the siRNA mixture. Cells were incubated for 24hours prior to RNA purification. Single dose experiments were performedat final concentrations of 10 nM and 0.1 nM. An additional concentrationof 0.01 nM was performed for selected duplexes. Final duplexconcentrations for dose response experiments were 10, 1.67, 0.278,0.046, 0.0077, 0.0012, 0.0002, and 0.000035 nM.

Endogenous System (Cynomolgus):

Transfection was carried out by adding 14.8 μl of Opti-MEM plus 0.2 μlof Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat#13778-150) to 5 μl of siRNA duplexes per well into a 96-well plate andincubated at room temperature for 15 minutes. Primary cynomolgushepatocytes (M003055-P, Celsis) were thawed and prepared in InVitroGROCP plating medium (Z99029, Celsis). 80 μl of complete growth mediawithout antibiotic containing ˜2×10⁴ cynomolgus hepatocytes were thenadded to the siRNA mixture. Cells were incubated for 24 hours prior toRNA purification. Single dose experiments were performed at finalconcentrations of 10 nM and 0.1 nM. Final duplex concentrations for doseresponse experiments were 10, 1.67, 0.278, 0.046, 0.0077, 0.0012,0.0002, and 0.000035 nM.

Dual Luciferase Assay (Promega Part E2980):

For cells transfected with the psiCHECK2 vector containing the humanHAMP ORF, the Dual Luciferase assay was performed to measure reductionin HAMP levels. Forty-eight hours after transfection, the media wasremoved over the cells, and cells received 150 uL of a 1:1 mixture ofcomplete growth medium and Dual-Glo Luciferase Reagent. As a control,these reagents were also added to empty wells; data derived from thesesamples were thus used as a blank measurement. Cells were then incubatedfor 30 minutes at room temperature on a shaker, protected from light. Atthis time, luminescence was determined using a SpectraMax M5 (MolecularDevices) with an integration time of 500 ms, and resulting data definedas the firefly luciferase signal. Following measurement, 754 of Dual-GloStop & Glo Reagent was added and the plates incubated in the dark atroom temperature, without shaking. After an additional 10 minutesluminescence was again measured as above, and resulting data defined asthe renilla luciferase signal. Data were background-subtracted, and therenilla values normalized to the firefly Luciferase values. Data werethen expressed as percent mock-transfected or percent AD-1955.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen, Part#: 610-12):

Cells were harvested and lysed in 150 μl of Lysis/Binding Buffer thenmixed for 5 minute at 850 rpm using an Eppendorf Thermomixer (the mixingspeed was the same throughout the process). Ten microliters of magneticbeads and 80 μl Lysis/Binding Buffer mixture were added to a roundbottom plate and mixed for 1 minute. Magnetic beads were captured usingmagnetic stand and the supernatant was removed without disturbing thebeads. After removing supernatant, the lysed cells were added to theremaining beads and mixed for 5 minutes. After removing supernatant,magnetic beads were washed 2 times with 150 μl Wash Buffer A and mixedfor 1 minute. Beads were capture again and supernatant removed. Beadswere then washed with 150 μl Wash Buffer B, captured and supernatant wasremoved. Beads were next washed with 150 μl Elution Buffer, captured andsupernatant removed. Beads were allowed to dry for 2 minutes. Afterdrying, 50 μl of Elution Buffer was added and mixed for 5 minutes at 70°C. Beads were captured on magnet for 5 minutes. 40 μl of supernatant wasremoved and added to another 96 well plate.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit(Applied Biosystems, Foster City, Calif., Cat #4368813):

A master mix of 2 μl 10× Buffer, 0.8 μl 25× dNTPs, 2 μl Random primers,1 μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.2 μl of H2O perreaction were added into 10 μl total RNA. cDNA was generated using aBio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through thefollowing steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C.hold.

Real Time PCR:

For human HAMP, 2 μl of cDNA were added to a master mix containing 0.5μl GAPDH TaqMan Probe (Applied Biosystems Cat #4326317E), 0.5 μl HAMPTaqMan probe (Applied Biosystems cat # Hs00221783_m1) and 5 μlLightcycler 480 probe master mix (Roche Cat #04887301001) per well in a384 well 50 plates (Roche cat #04887301001). For cynomolgus HAMP, 2 μlof cDNA were added to a master mix containing 0.5 μl 18 s TaqMan Probe(Applied Biosystems Cat #4319413E), 0.1 μl 10× custom cynomolgus HAMPprobe (Forward primer: CTCCGTTTTCCCACAACA (SEQ ID NO: 39); Reverseprimer: CAGCACATCCCACACTTT (SEQ ID NO: 40); Probe: ACCCACTTCCCCATCTGCATT(SEQ ID NO: 41)), and 5 μl Lightcycler 480 probe master mix (Roche Cat#04887301001) per well in a 384 well 50 plates (Roche cat #04887301001).Real time PCR was done in an ABI 7900HT Real Time PCR system (AppliedBiosystems) using the ΔΔCt(RQ) assay. Each duplex was tested in twoindependent transfections and each transfection was assayed induplicate, unless otherwise noted in the summary tables.

To calculate relative fold change, real time data were analyzed usingthe ΔΔCt method and normalized to assays performed with cellstransfected with 10 nM AD-1955, or mock transfected cells. IC50s werecalculated using a 4 parameter fit model using XLFit and normalized tocells transfected with 10 nM AD-1955, mock transfected, or to theaverage lowest dose.

Table 6 shows the HAMP single dose screening data of the modifiedduplexes using the dual luciferase assay. Data are expressed as apercent of mock or AD-1955.

Table 7 shows the HAMP single dose screening data of the unmodifiedduplexes using the human endogenous assay. Data are expressed as apercent of mock.

Table 8 shows the HAMP single dose screening data of the modifiedduplexes using the human endogenous assay. Data are expressed as apercent of mock.

Table 9 shows the HAMP dose response data of modified and unmodifiedduplexes using the dual luciferase assay. Cells used included HepG2 andCyno primary hepatocytes.

Example 4 HFE2 siRNA Design

siRNA design was carried out to identify siRNAs targeting human, rhesus(Macaca mulatta), mouse, and rat HFE2 transcripts annotated in the NCBIGene database website noted above. There are at least 4 annotated humanHFE2 transcripts and at least 3 annotated rhesus transcripts.Accordingly, we focused on the shortest annotated transcript for human,and the rhesus transcript which shared the greatest number oforthologous human exons, and designed on sequences held in common by thealternate transcripts. Design used the following transcripts from theNCBI RefSeq collection: Human—NM_213652.3; Rhesus—XM_001092987.1;Mouse—NM_027126.4; Rat—NM_001012080.1. Due to high primate/rodentsequence divergenge, siRNA duplexes were designed in two separatebatches. The first batch matched human and rhesus; the second matchedmouse and rat. All siRNA duplexes were designed that shared 100%identity with all listed human/rhesus or mouse/rat transcripts.

siRNA Design, Specificity, and Efficacy Prediction

The predicted specificity of all possible 19mers was predicted from eachsequence. Candidate 19mers were selected that lacked repeats longer than7 nucleotides. These siRNAs were used in comprehensive searches againstthe appropriate transcriptomes.

siRNAs strands were assigned to a category of specificity according tothe calculated scores: a score above 3 qualifies as highly specific,equal to 3 as specific and between 2.2 and 2.8 as moderately specific.We sorted by the specificity of the antisense strand. We then selectedduplexes whose antisense oligos lacked GC at the first position, lackedG at both positions 13 and 14, and had 4 or more Us or As in the seedregion.

siRNA Sequence Selection

A total of 47 sense and 47 antisense derived human/rhesus, and 40 senseand 40 antisense derived mouse/rat siRNA oligos were synthesized andformed into duplexes.

Table 10A provides the sequences of the sense and antisense strands ofthe duplexes targeting the HFE2 gene.

Example 5 TFR2 siRNA Design Transcripts

siRNA design was carried out to identify siRNAs targeting human, rhesus(Macaca mulatta), mouse, and rat TFR2 transcripts annotated in the NCBIGene database website noted above. Design used the following transcriptsfrom the NCBI RefSeq collection: Human—NM_003227.3, NM_001206855.1;Rhesus—XM_001113151.2; Mouse—NM_015799.3; Rat—NM_001105916.1. Due tohigh primate/rodent sequence divergenge, siRNA duplexes were designed inthree separate batches. The first batch matched human and rhesus; thesecond matched human, rhesus, and mouse; the last batch matched mouseand rat. All siRNA duplexes were designed that shared 100% identity withall listed human/rhesus, human/rhesus/mouse, or mouse/rat transcripts.

siRNA Design, Specificity, and Efficacy Prediction

The predicted specificity of all possible 19mers was predicted from eachsequence. Candidate 19mers were selected that lacked repeats longer than7 nucleotides. These siRNAs were used in comprehensive searches againstthe appropriate transcriptomes.

siRNAs strands were assigned to a category of specificity according tothe calculated scores: a score above 3 qualifies as highly specific,equal to 3 as specific and between 2.2 and 2.8 as moderately specific.We sorted by the specificity of the antisense strand. We then selectedduplexes whose antisense oligos lacked GC at the first position, lackedG at both positions 13 and 14, and had 3 or more Us or As in the seedregion.

siRNA Sequence Selection

A total of 40 sense and 40 antisense derived human/rhesus, 5 sense and 5antisense derived human/rhesus/mouse, and 45 sense and 45 antisensederived mouse/rat siRNA oligos were synthesized and formed intoduplexes.

Table 10B provides the sequences of the sense and antisense strands ofthe duplexes targeting the TFR2 gene.

Example 6 HFE2 and TFR2 siRNA Screening Cell Culture and Transfections:

Endogenous system (Human): For TFR2, HepG2 cells were used; Hep3b cellswere used for HFE2. HepG2 and Hep3b cells (ATCC, Manassas, Va.) weregrown to near confluence at 37° C. in an atmosphere of 5% CO₂ in MEM(Gibco) supplemented with 10% FBS before being released from the plateby trypsinization. Transfection was carried out by adding 14.8 μl ofOpti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen,Carlsbad Calif. cat #13778-150) to 5 μl of siRNA duplexes per well intoa 96-well plate and incubated at room temperature for 15 minutes. 80 μlof complete growth media without antibiotic containing ˜2×10⁴ HepG2cells were then added to the siRNA mixture. Cells were incubated for 24hours prior to RNA purification. Single dose experiments were performedat final concentrations of 10 nM and 0.1 nM. An additional concentrationof 0.01 nM was performed for selected duplexes. Final duplexconcentrations for dose response experiments were 10, 1.67, 0.278,0.046, 0.0077, 0.0012, 0.0002, and 0.000035 nM.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen, Part#: 610-12):

Cells were harvested and lysed in 150 μl of Lysis/Binding Buffer thenmixed for 5 minute at 850 rpm using an Eppendorf Thermomixer (the mixingspeed was the same throughout the process). Ten microliters of magneticbeads and 80 μl Lysis/Binding Buffer mixture were added to a roundbottom plate and mixed for 1 minute. Magnetic beads were captured usingmagnetic stand and the supernatant was removed without disturbing thebeads. After removing supernatant, the lysed cells were added to theremaining beads and mixed for 5 minutes. After removing supernatant,magnetic beads were washed 2 times with 150 μl Wash Buffer A and mixedfor 1 minute. Beads were capture again and supernatant removed. Beadswere then washed with 150 μl Wash Buffer B, captured and supernatant wasremoved. Beads were next washed with 150 μl Elution Buffer, captured andsupernatant removed. Beads were allowed to dry for 2 minutes. Afterdrying, 50 μl of Elution Buffer was added and mixed for 5 minutes at 70°C. Beads were captured on magnet for 5 minutes. 40 μl of supernatant wasremoved and added to another 96 well plate.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit(Applied Biosystems, Foster City, Calif., Cat #4368813):

A master mix of 2 μl 10× Buffer, 0.8 μl 25× dNTPs, 2 μl Random primers,1 μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.2 μl of H2O perreaction were added into 10 μl total RNA. cDNA was generated using aBio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through thefollowing steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C.hold.

Real Time PCR:

2 μl of cDNA were added to a master mix containing 0.5 μl GAPDH TaqManProbe (Applied Biosystems Cat #4326317E), 0.5 μl HFE2 or TFR2 probes,and 5 μl Lightcycler 480 probe master mix (Roche Cat #04887301001) perwell in a 384 well 50 plates (Roche cat #04887301001). HFE2 and TFR2probes were Applied Biosystems cat # Hs02378779_s1 and Hs00162690_m1,respectively. Real time PCR was done in an ABI 7900HT Real Time PCRsystem (Applied Biosystems) using the ΔΔCt(RQ) assay. Each duplex wastested in two independent transfections and each transfection wasassayed in duplicate, unless otherwise noted in the summary tables.

To calculate relative fold change, real time data were analyzed usingthe ΔΔCt method and normalized to assays performed with cellstransfected with 10 nM AD-1955, or mock transfected cells. IC50s werecalculated using a 4 parameter fit model using XLFit and normalized tocells transfected with 10 nM AD-1955, mock transfected, or to theaverage lowest dose.

Table 11 shows the HFE2 and TFR2 single dose screening data of theduplexes using the human endogenous assay.

Table 12 shows the HFE2 and TFR2 dose response data of the duplexes.

Example 7 HFE siRNA Design Transcripts

siRNA design was carried out to identify siRNAs targeting human, rhesus(Macaca mulatta), mouse, and rat HFE transcripts annotated in the NCBIGene database website noted above. There are at least 9 annotated humanHFE transcripts, at least 5 annotated rhesus transcripts, and at least 4annotated rat transcripts. Accordingly, we focused on the shortestannotated transcripts for human, rhesus, and rat HFE, and designed onsequences held in common by the alternate transcripts. Design used thefollowing transcripts from the NCBI RefSeq collection:Human—NM_139006.2; Rhesus—XM_001085598.2; Mouse—NM_010424.4;Rat—NM_001173435.1. Due to high primate/rodent sequence divergenge,siRNA duplexes were designed in two separate batches. The first batchmatched human and rhesus; the second matched mouse and rat. All siRNAduplexes were designed that shared 100% identity with all listedhuman/rhesus or mouse/rat transcripts.

siRNA Design, Specificity, and Efficacy Prediction

The predicted specificity of all possible 19mers was predicted from eachsequence. Candidate 19mers were selected that lacked repeats longer than7 nucleotides. These siRNAs were used in comprehensive searches againstthe appropriate transcriptomes.

siRNAs strands were assigned to a category of specificity according tothe calculated scores: a score above 3 qualifies as highly specific,equal to 3 as specific and between 2.2 and 2.8 as moderately specific.We sorted by the specificity of the antisense strand. We then selectedduplexes whose antisense oligos lacked GC at the first position, lackedG at both positions 13 and 14, and had 3 or more Us or As in the seedregion.

siRNA Sequence Selection

A total of 46 sense and 46 antisense derived human/rhesus, and 24 senseand 24 antisense derived mouse/rat siRNA oligos are synthesized andformed into duplexes. The duplexes are screened using the methodsdescribed above. One or more duplexes are selected for further testing.

Example 8 BMPR1a siRNA Design Transcripts

siRNA design was carried out to identify siRNAs targeting mouse and ratBMPR1A transcripts annotated in the NCBI Gene database website notedabove. Design used the following transcripts from the NCBI RefSeqcollection: Mouse—NM_009758.4; Rat—NM_030849.1. All siRNA duplexes weredesigned that shared 100% identity with all listed mouse/rattranscripts.

siRNA Design, Specificity, and Efficacy Prediction

The predicted specificity of all possible 19mers was predicted from eachsequence. Candidate 19mers were selected that lacked repeats longer than7 nucleotides. These siRNAs were used in comprehensive searches againstthe appropriate transcriptomes.

siRNAs strands were assigned to a category of specificity according tothe calculated scores: a score above 3 qualifies as highly specific,equal to 3 as specific and between 2.2 and 2.8 as moderately specific.We sorted by the specificity of the antisense strand. We then selectedduplexes whose antisense oligos lacked GC at the first position, lackedG at both positions 13 and 14, and had 4 or more Us or As in the seedregion.

siRNA Sequence Selection

A total of 46 sense and 46 antisense derived mouse/rat siRNA oligos aresynthesized and formed into duplexes. The duplexes are screened usingthe methods described above. One or more duplexes are selected forfurther testing.

Example 9 SMAD4 siRNA Design Transcripts

siRNA design was carried out to identify siRNAs targeting human andmouse SMAD4 transcripts annotated in the NCBI Gene database websitenoted above. Design used the following transcripts from the NCBI RefSeqcollection: Human—NM_005359.5; Mouse—NM_008540.2. All siRNA duplexeswere designed that shared 100% identity with all listed human/mousetranscripts.

siRNA Design, Specificity, and Efficacy Prediction

The predicted specificity of all possible 19mers was predicted from eachsequence. Candidate 19mers were selected that lacked repeats longer than7 nucleotides. These siRNAs were used in comprehensive searches againstthe appropriate transcriptomes.

siRNAs strands were assigned to a category of specificity according tothe calculated scores: a score above 3 qualifies as highly specific,equal to 3 as specific and between 2.2 and 2.8 as moderately specific.We sorted by the specificity of the antisense strand. We then selectedduplexes whose antisense oligos lacked GC at the first position, lackedG at both positions 13 and 14, and had 4 or more Us or As in the seedregion.

siRNA Sequence Selection

Tables 15-16 provide the sequences of the sense and antisense strands ofthe duplexes targeting SMAD4 mRNA at the indicated locations. Someduplexes were modified as indicated. These siRNA oligos were synthesizedand formed into duplexes for further testing as described below.

Example 10 IL6R siRNA Design Transcripts

siRNA design was carried out to identify siRNAs targeting mouse and ratIL6R transcripts annotated in the NCBI Gene database website notedabove. Design used the following transcripts from the NCBI RefSeqcollection: Mouse—NM_010559.2; Rat—NM_017020.3. All siRNA duplexes weredesigned that shared 100% identity with all listed mouse/rattranscripts.

siRNA Design, Specificity, and Efficacy Prediction

The predicted specificity of all possible 19mers was predicted from eachsequence. Candidate 19mers were selected that lacked repeats longer than7 nucleotides. These siRNAs were used in comprehensive searches againstthe appropriate transcriptomes.

siRNAs strands were assigned to a category of specificity according tothe calculated scores: a score above 3 qualifies as highly specific,equal to 3 as specific and between 2.2 and 2.8 as moderately specific.We sorted by the specificity of the antisense strand. We then selectedduplexes whose antisense oligos lacked GC at the first position and had2 or more Us or As in the seed region.

siRNA Sequence Selection

A total of 44 sense and 44 antisense derived mouse/rat siRNA oligos aresynthesized and formed into duplexes. The duplexes are screened usingthe methods described above. One or more duplexes are selected forfurther testing.

Example 11 BMP6 siRNA Design Transcripts

siRNA design was carried out to identify siRNAs targeting human, rhesus(Macaca mulatta), mouse, and rat BMP6 transcripts annotated in the NCBIGene database website noted above. Design used the following transcriptsfrom the NCBI RefSeq collection: Human—NM_001718.4;Rhesus—XM_001085364.2; Mouse—NM_007556.2; Rat—NM_013107.1. Due to highprimate/rodent sequence divergenge, siRNA duplexes were designed inthree separate batches. The first batch matched human and rhesus; thesecond matched human, rhesus, and mouse; the last batch matched mouseand rat. All siRNA duplexes were designed that shared 100% identity withall listed human/rhesus, human/rhesus/mouse, or mouse/rat transcripts.

siRNA Design, Specificity, and Efficacy Prediction

The predicted specificity of all possible 19mers was predicted from eachsequence. Candidate 19mers were selected that lacked repeats longer than7 nucleotides. These siRNAs were used in comprehensive searches againstthe appropriate transcriptomes.

siRNAs strands were assigned to a category of specificity according tothe calculated scores: a score above 3 qualifies as highly specific,equal to 3 as specific and between 2.2 and 2.8 as moderately specific.We sorted by the specificity of the antisense strand. We then selectedduplexes whose antisense oligos lacked GC at the first position, lackedG at both positions 13 and 14, and had 3 or more Us or As in the seedregion.

siRNA Sequence Selection

Table 21 provides the sequences of the sense and antisense strands ofthe duplexes targeting BMP6 mRNA. Some duplexes were modified asindicated. These siRNA oligos were synthesized and formed into duplexesfor further testing using the methods described herein.

Example 12 Neo1 siRNA Design Transcripts

siRNA design was carried out to identify siRNAs targeting human andmouse NEO1 transcripts annotated in the NCBI Gene database website notedabove. There are 2 annotated mouse NEO1 transcripts. Accordingly, wefocused on the shortest annotated transcripts for mouse NEO1, anddesigned on sequences held in common by the alternate transcripts.Design used the following transcripts from the NCBI RefSeq collection:Human—NM_002499.2; Mouse—NM_001042752.1. All siRNA duplexes weredesigned that shared 100% identity with all listed human/mousetranscripts.

siRNA Design, Specificity, and Efficacy Prediction

The predicted specificity of all possible 19mers was predicted from eachsequence. Candidate 19mers were selected that lacked repeats longer than7 nucleotides. These siRNAs were used in comprehensive searches againstthe appropriate transcriptomes.

siRNAs strands were assigned to a category of specificity according tothe calculated scores: a score above 3 qualifies as highly specific,equal to 3 as specific and between 2.2 and 2.8 as moderately specific.We sorted by the specificity of the antisense strand. We then selectedduplexes whose antisense oligos lacked GC at the first position, lackedG at both positions 13 and 14, and had 3 or more Us or As in the seedregion.

siRNA Sequence Selection

Tables 17-18 provide the sequences of the sense and antisense strands ofthe duplexes targeting NEO1 mRNA at the indicated locations. Someduplexes were modified as indicated. These siRNA oligos were synthesizedand formed into duplexes for further testing as described below.

Example 13 Activity of Murine siRNA In Vivo

The efficacy of one or more siRNAs described above is determined inmice, e.g., normal 10 week old 129s6/svEvTac mice using AD-1955targeting luciferase as a control. The siRNAs are formulated asdescribed herein and administered, e.g., through i.v. bolus at a doseof, e.g., 10 mg/kg. Forty eight hours after injection, the liver andserum samples are harvested. The liver mRNA levels of the target mRNAare determined by qRT-PCR using gene specific primers and serum ironlevels are determined using Feroxcine (Randox Life Sciences) and Hitachi717 instrument.

siRNAs that result in lowering of mRNA are selected for furtherevaluation.

Example 14 Activity of Murine Hepcidin siRNA In Vivo

The efficacy of an HAMP siRNA AD-10812 was determined in mice usingAF-011 formulated control siRNA and PBS as controls. Each siRNA wasformulated with AF-011. AF-011 is also known as LNP11 (See Table Aabove; MC-3/DSPC/Cholesterol/PEG-DMG (50/10/38.5/1.5); Lipid:siRNA10:1)).

position in mouse SEQ SEQ access. # sense strand sequence ID antisensestrand ID duplex NM_032541.1 (5′-3′) NO sequence (5′-3′) NO name 245-263uGcuGuAAcAAuucccAGuTsT 42 ACUGGGAAUUGUuAcAGcATsT 43 AD-10812

PBS and the siRNAs were administered at various dosages to the mice asshown in FIG. 1: 1 mg/kg, 0.3 mg/kg, 0.1 mg/kg, 0.03 mg/kg, 0.01 mg/kg,and 0.003 mg/kg. A single siRNA dose was administered to each mouse.After injection, liver and serum samples were harvested from the mice.The liver Hamp1 mRNA levels were determined by qRT-PCR using Hamp1specific primers and serum iron levels were determined FIG. 1 shows theHAMP1 mRNA levels in mouse liver following various dosages of siRNA andthe serum iron concentration (μg/dL) following various dosages of siRNA.

Administration of AD-10812 HAMP siRNA to mice resulted in lowering ofHAMP mRNA by >80% following a single dose. Administration of AD-10812HAMP siRNA to mice resulted in an approximately 2-fold increase in serumiron following a single dose.

Example 15 Activity of Hepcidin siRNA in Nonhuman Primates (NHPs) InVivo

The efficacy of an HAMP siRNA AD-11459 was determined in male cynomolgusmonkeys using AF-011 siRNA as a control.

Duplex Start Sense SEQ ID Antisense Target ID Position Name SenseSequence NO Name Antisense Sequence SEQ ID NO HAMP AD- 382 A-18280.2GAAcAuAGGucuuG 30 A-18304.1 uAuUCcAAGACCuAuGuUC 44 11459 GAAuAdTsdTdTsdT

Each siRNA was formulated with AF-011. The siRNAs were administeredintravenously at a dose of 1 mg/kg via a 15 minute infusion. A singlesiRNA dose was administered to each monkey. After injection, liver andserum samples were harvested. Liver samples were taken at 48 hours (h)post-injection. Serum samples were taken at Day −9, Day −6, Day −3, 24 hpost-injection, and 48 h post-injection. The liver Hamp mRNA levels weredetermined by qRT-PCR using Hamp specific primers and serum iron levelswere determined Serum HAMP protein levels were also determined. FIG. 2shows the HAMP mRNA levels in liver following siRNA administration aswell as the serum iron concentration (μg/dL) and the HAMP serum proteinconcentration (mg/mL) following siRNA administration.

Single administration of LNP-siRNA AD-11459 resulted in rapid reductionof hepcidin mRNA and protein levels and elevation of serum iron levelsin NHPs.

Example 16 Silencing of Murine TFR2 Via siRNA In Vivo

The efficacy of TFR2 siRNA AD-47882 (see table below for sequences) wasdetermined in C57BL6 mice using AF-011 siRNA and PBS as controls. EachsiRNA was formulated with AF-011. PBS and the siRNAs were administeredat various dosages to the mice as shown in FIG. 3: 1 mg/kg, 0.3 mg/kg,0.1 mg/kg, and 0.03 mg/kg. After injection, liver and serum samples wereharvested from the mice. The liver Hamp1 and TFR2 mRNA levels weredetermined by qRT-PCR using gene specific primers and transferrinsaturation were determined at 48 hours post-injection. FIG. 3 shows theHAMP1 and TFR2 mRNA levels in mouse liver following various dosages ofsiRNA and the percent (%) transferrin saturation following variousdosages of siRNA.

Duplex Target ID Sense Sequence SEQ ID NO Antisense Sequence SEQ ID NOTFR2 AD- ccAcGuGAuucuccu 45 AGAAAGGAGAAUcACG 46 47882 uucudTsdT UGGdTsdT

Administration of AD-47882 siRNA to mice resulted in lowering of HAMPand TFR2 mRNA levels. Administration of AD-47882 siRNA to mice resultedin an increase in transferrin saturation.

Example 17 Silencing of Murine TFR2 Via siRNA In Vivo

The duration of TFR2 siRNA AD-47882 was determined in C57BL6 mice. ThesiRNAs were administered at in a single 0.3 mg/kg dose intravenously.Each siRNA was formulated with AF-011. After injection, liver and serumsamples were harvested from the mice at various time points shown inFIG. 4. The liver Hamp1 and TFR2 mRNA levels were determined by qRT-PCRusing gene specific primers and transferrin saturation were determinedFIG. 4 shows the HAMP1 and TFR2 mRNA levels in mouse liver followingadministration of siRNA and the percent (%) transferrin saturation overa 30 day time course.

Administration of AD-47882 siRNA to mice resulted in lowering of HAMPand TFR2 mRNA levels. Administration of AD-47882 siRNA to mice resultedin an increase in transferrin saturation.

Example 18 Silencing of Rat TFR2 Via siRNA In Vivo

The duration and efficacy of TFR2 siRNA AD-47882 was determined in maleLewis rats using the anemia of chronic disease (ACD) model described inCoccia et al., Exp. Hematology, 2001. Briefly, anemia was initiated inthe rats with a single intraperaoneal (i.p.) injection of PG-APS(polymers from Group A Streptococci). The rats were then treated 3× perweek with AD-47882 siRNA, AF-011 control siRNA, or saline controlstarting at day 21 post PG-APS. Each siRNA was formulated with AF-011.Serum and hematology parameters were measured biweekly and at 48 hourspost final treatment. Serum samples were harvested from the rats atvarious time points as shown in FIG. 5. Liver mRNA measurement was takenat 48 hours post final treatment. FIG. 5 shows the HAMP1 and TFR2 mRNAlevels in rat liver following administration of siRNA. FIG. 5 also showsthe serum iron and Hb concentrations at various time points.

Administration of AD-47882 siRNA resulted in lowering of HAMP and TFR2mRNA levels. Administration of AD-47882 siRNA resulted in an approximate2× increase in serum iron upon treatment. Administration of AD-47882siRNA resulted in an increase in Hb levels to 11-12 g/dL with treatment.

Example 19 TFR2 siRNA Selection and Screening

siRNA Sequence Selection

Table 13 provides the sequences of the sense and antisense strands ofthe duplexes targeting the TFR2 gene at the indicated locations (64 or239). These siRNA oligos were synthesized and formed into duplexes.

Cell Culture and Transfections:

Endogenous system (Human): For TFR2, HepG2 cells were used. HepG2 cells(ATCC, Manassas, Va.) were grown to near confluence at 37° C. in anatmosphere of 5% CO2 in MEM (Gibco) supplemented with 10% FBS beforebeing released from the plate by trypsinization. Transfection wascarried out by adding 14.8 μl of Opti-MEM plus 0.2 μl of LipofectamineRNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl ofsiRNA duplexes per well into a 96-well plate and incubated at roomtemperature for 15 minutes. 80 μl of complete growth media withoutantibiotic containing ˜2×104 HepG2 cells were then added to the siRNAmixture. Cells were incubated for 24 hours prior to RNA purification.Single dose experiments were performed at final concentrations of 10 nMand 0.1 nM and 0.01 nM. An additional concentration of 0.01 nM wasperformed for selected duplexes.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen, Part#: 610-12):

Cells were harvested and lysed in 150 μl of Lysis/Binding Buffer thenmixed for 5 minute at 850 rpm using an Eppendorf Thermomixer (the mixingspeed was the same throughout the process). Ten microliters of magneticbeads and 80 μl Lysis/Binding Buffer mixture were added to a roundbottom plate and mixed for 1 minute. Magnetic beads were captured usingmagnetic stand and the supernatant was removed without disturbing thebeads. After removing supernatant, the lysed cells were added to theremaining beads and mixed for 5 minutes. After removing supernatant,magnetic beads were washed 2 times with 150 μl Wash Buffer A and mixedfor 1 minute. Beads were capture again and supernatant removed. Beadswere then washed with 150 μl Wash Buffer B, captured and supernatant wasremoved. Beads were next washed with 150 μl Elution Buffer, captured andsupernatant removed. Beads were allowed to dry for 2 minutes. Afterdrying, 50 μl of Elution Buffer was added and mixed for 5 minutes at 70°C. Beads were captured on magnet for 5 minutes. 40 μl of supernatant wasremoved and added to another 96 well plate.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit(Applied Biosystems, Foster City, Calif., Cat #4368813):

A master mix of 2 μl 10× Buffer, 0.8 μl 25× dNTPs, 2 μl Random primers,1 μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.2 μl of H2O perreaction were added into 10 μl total RNA. cDNA was generated using aBio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through thefollowing steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C.hold.

Real Time PCR:

2 μl of cDNA were added to a master mix containing 0.5 μl GAPDH TaqManProbe (Applied Biosystems Cat #4326317E), 0.5 μl TFR2 probes, and 5 μlLightcycler 480 probe master mix (Roche Cat #04887301001) per well in a384 well 50 plates (Roche cat #04887301001). TFR2 probes were AppliedBiosystems cat # Hs02378779_s1 and Hs00162690_m1, respectively. Realtime PCR was done in an ABI 7900HT Real Time PCR system (AppliedBiosystems) using the ΔΔCt(RQ) assay. Each duplex was tested in twoindependent transfections and each transfection was assayed induplicate, unless otherwise noted in the summary tables.

To calculate relative fold change, real time data were analyzed usingthe ΔΔCt method and normalized to assays performed with cellstransfected with 10 nM AD-1955, or mock transfected cells. IC50s werecalculated using a 4 parameter fit model using XLFit and normalized tocells transfected with 10 nM AD-1955, mock transfected, or to theaverage lowest dose.

Table 14 shows the TFR2 dose response data of the duplexes.

Example 20 Activity of TFR2 and HAMP siRNA in Non-Human Primates (NHPs)In Vivo

The efficacy of AD-52590, AD-51707, and AD-48141 was determined inseparate cynomolgus monkeys (3 each) using PBS as a control. Thesequence of AD-52590, AD-51707, and AD-48141 are shown below and inTable 4, 10B, and 13.

Duplex Start SEQ ID Target ID Position SEQ ID NO Sense Sequence NOAntisense Sequence TFR2 AD- 239 35 cAGGcAGcCAAAcCuCAuUdTsdT 38AAUGAGGUuUGGCUGcCugdTsdT 52590 TFR2 AD- 1051 47 ccuucAAucAAAcccAGuudTsdT48 AACuGGGUuUGAuUGAAGGdTsdT 51707 HAMP AD- 382 30GAAcAuAGGucuuGGAAuAdTdT 44 UAuUCcAAGACCuAuGuUCdTdT 48141

Each siRNA was formulated with AF-011. The siRNAs were administeredintravenously as indicated (0.1 mg/kg, 0.03 mg/kg, or 1 mg/kg) via a 15minute infusion. A single siRNA dose was administered to each monkey.After injection, liver and serum samples were harvested. Liver biopsysamples were taken at 48 hours (h) post-injection. Serum samples weretaken at Day −9, Day −6, Day −3, 24 h post-injection, and 48 hpost-injection. The liver Hamp mRNA levels were determined by qRT-PCRusing Hamp specific primers. The liver TFR2 mRNA levels were determinedby qRT-PCR using TFR2 specific primers. Serum iron levels weredetermined and are shown in μg/dL. Serum HAMP protein levels were alsodetermined and are shown in ng/mL.

FIG. 6 shows HAMP mRNA levels in the liver of each animal followingsiRNA administration, relative to PBS controls. FIG. 7 shows TFR2 mRNAlevels in the liver of each animal following siRNA administration,relative to PBS controls. FIG. 8 shows that serum iron concentration wasincreased in each animal after 1 mg/kg AD-52590 siRNA administration.FIG. 9 shows that the HAMP serum protein concentration was decreased ineach animal following 1 mg/kg AD-52590 siRNA administration.

Single administration of AD-52590 resulted in rapid reduction ofhepcidin mRNA and protein levels, TFR2 mRNA levels, and elevation ofserum iron levels in NHPs.

Example 21 NEO1 and SMAD4 Duplex Screening

Human/mouse cross-reactive Neo1 and Smad4 siRNAs were screened inprimary mouse hepatocytes. Duplexes are shown in Tables 15, 16, 17, and18.

Cell Culture and Transfections:

Freshly isolated primary mouse hepatocytes (PMH) were transfected byadding 14.8 μl of Opti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well(Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl of siRNA duplexesper well into a 96-well plate and incubated at room temperature for 15minutes. 80 μl of primary hepatocyte media containing ˜2×10⁴ PMH cellswere then added to the siRNA mixture. Cells were incubated for either 24prior to RNA purification. Single dose experiments were performed at 10nM and 0.1 nM final duplex concentration.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen, Part#: 610-12):

Cells were harvested and lysed in 150 μl of Lysis/Binding Buffer thenmixed for 5 minute at 850 rpm using an Eppendorf Thermomixer (the mixingspeed was the same throughout the process). Ten microliters of magneticbeads and 80 μl Lysis/Binding Buffer mixture were added to a roundbottom plate and mixed for 1 minute. Magnetic beads were captured usingmagnetic stand and the supernatant was removed without disturbing thebeads. After removing supernatant, the lysed cells were added to theremaining beads and mixed for 5 minutes. After removing supernatant,magnetic beads were washed 2 times with 150 μl Wash Buffer A and mixedfor 1 minute. Beads were capture again and supernatant removed. Beadswere then washed with 150 μl Wash Buffer B, captured and supernatant wasremoved. Beads were next washed with 150 μl Elution Buffer, captured andsupernatant removed. Beads were allowed to dry for 2 minutes. Afterdrying, 50 μl of Elution Buffer was added and mixed for 5 minutes at 70°C. Beads were captured on magnet for 5 minutes. 40 μl of supernatant wasremoved and added to another 96 well plate.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit(Applied Biosystems, Foster City, Calif., Cat #4368813):

A master mix of 2 μl 10× Buffer, 0.8 μl 25× dNTPs, 2 μl Random primers,1 μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.2 μl of H2O perreaction were added into 10 μl total RNA. cDNA was generated using aBio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through thefollowing steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C.hold.

Real Time PCR:

2 μl of cDNA were added to a master mix containing 0.5 μl of mouse GAPDHTaqMan Probe (Applied Biosystems Cat #4352932E), 0.5 μl Neo1 or SMAD4TaqMan probe (Applied Biosystems cat # Neo1-Mm00476326_m1 or SMAD4Mm03023996_m1) and 5 μl Lightcycler 480 probe master mix (Roche Cat#04887301001) per well in a 384 well 50 plates (Roche cat #04887301001).Real time PCR was done in an ABI 7900HT Real Time PCR system (AppliedBiosystems) using the ΔΔCt(RQ) assay. Each duplex was tested in twoindependent transfections and each transfection was assayed induplicate, unless otherwise noted.

To calculate relative fold change, real time data were analyzed usingthe ΔΔCt method and normalized to assays performed with cellstransfected with 10 nM AD-1955, or mock transfected cells. IC50s werecalculated using a 4 parameter fit model using XLFit and normalized tocells transfected with AD-1955 or naïve cells over the same dose range,or to its own lowest dose.

Table 19 shows the percent remaining mRNA remaining for each SMAD4duplex tested at 0.1 nM and 10 nM. Controls were 10 nM AD-1955, mocktransfected. Table 20 shows the percent remaining mRNA remaining foreach NEO1 duplex tested at 0.1 nM and 10 nM. Controls were 10 nMAD-1955, mock transfected.

Example 22 In Vivo Combinatorial Use of dsRNAs Targeting HAMP-RelatedmRNAs

The efficacy of TFR2 siRNA AD-47882 and HFE siRNA AD-47320 (see tablebelow for sequences) alone and in combination was determined in C57BL6female mice using AF-011-Luc siRNA and PBS as controls. Each siRNA wasformulated with AF-011. PBS and the siRNAs were administered at various(mg/kg) dosages to the mice as shown on the X-axis of each subfigure(A-D) of FIG. 10. 48 hours after injection, liver and serum samples wereharvested from the mice.

Duplex Target ID Accession Number Sense Sequence SEQ ID NO AntisenseSequence SEQ ID NO HFE AD- NM_010424.4 uuuucuccAGuuAAG 49UGAACUuAACUGGAGA 51 47320 uucAdTsdT AAAdTsdT HFE Unmod NM_010424.4UUUUCUCCAGUUA 50 UGAACUUAACUGGAG 52 AD- AGUUCA AAAA 47320

The liver Hamp1, HFE, and TFR2 mRNA levels were determined by qRT-PCRusing gene specific primers. Blood was processed into serum to measureserum iron, transferrin saturation, and UIBC. FIG. 10A shows the HAMP1,HFE, and TFR2 mRNA levels in mouse liver following various dosages ofeach siRNA group or PBS. FIGS. 10B-D shows serum iron concentration,transferrin saturation, and UIBC concentration in the serum of eachgroup tested.

Example 23 Inhibition of HAMP in Humans

A human subject is treated with a siRNA targeted to a HAMP gene toinhibit expression of the HAMP gene to treat a condition. In someinstances, one or more additional siRNAs are co-administered, e.g., ansiRNA targeted to a HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/orNEO1 gene.

A subject in need of treatment is selected or identified.

The identification of the subject can occur in a clinical setting, orelsewhere, e.g., in the subject's home through the subject's own use ofa self-testing kit.

At time zero, a suitable first dose of an siRNA is administered to thesubject. The siRNA is formulated as described herein. After a period oftime following the first dose, e.g., 7 days, 14 days, and 21 days, thesubject's condition is evaluated. This measurement can be accompanied bya measurement of target gene expression in said subject, and/or theproducts of the successful siRNA-targeting of mRNA. Other relevantcriteria can also be measured. The number and strength of doses areadjusted according to the subject's needs.

Example 24 Inhibition of HFE2 in Humans

A human subject is treated with a siRNA targeted to a gene to inhibitexpression of the HFE2 gene to treat a condition. In some instances, oneor more additional siRNAs are co-administered, e.g., an siRNA targetedto a HAMP, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene.

A subject in need of treatment is selected or identified.

The identification of the subject can occur in a clinical setting, orelsewhere, e.g., in the subject's home through the subject's own use ofa self-testing kit.

At time zero, a suitable first dose of an siRNA is administered to thesubject. The siRNA is formulated as described herein. After a period oftime following the first dose, e.g., 7 days, 14 days, and 21 days, thesubject's condition is evaluated. This measurement can be accompanied bya measurement of target gene expression in said subject, and/or theproducts of the successful siRNA-targeting of mRNA. Other relevantcriteria can also be measured. The number and strength of doses areadjusted according to the subject's needs.

Example 25 Inhibition of HFE in Humans

A human subject is treated with a siRNA targeted to a gene to inhibitexpression of the HFE gene to treat a condition. In some instances, oneor more additional siRNAs are co-administered, e.g., an siRNA targetedto a HAMP, HFE2, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene.

A subject in need of treatment is selected or identified.

The identification of the subject can occur in a clinical setting, orelsewhere, e.g., in the subject's home through the subject's own use ofa self-testing kit.

At time zero, a suitable first dose of an siRNA is administered to thesubject. The siRNA is formulated as described herein. After a period oftime following the first dose, e.g., 7 days, 14 days, and 21 days, thesubject's condition is evaluated. This measurement can be accompanied bya measurement of target gene expression in said subject, and/or theproducts of the successful siRNA-targeting of mRNA. Other relevantcriteria can also be measured. The number and strength of doses areadjusted according to the subject's needs.

Example 26 Inhibition of TFR2 in Humans

A human subject is treated with a siRNA targeted to a gene to inhibitexpression of the TFR2 gene to treat a condition. In some instances, oneor more additional siRNAs are co-administered, e.g., an siRNA targetedto a HAMP, HFE2, HFE, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene.

A subject in need of treatment is selected or identified.

The identification of the subject can occur in a clinical setting, orelsewhere, e.g., in the subject's home through the subject's own use ofa self-testing kit.

At time zero, a suitable first dose of an siRNA is administered to thesubject. The siRNA is formulated as described herein. After a period oftime following the first dose, e.g., 7 days, 14 days, and 21 days, thesubject's condition is evaluated. This measurement can be accompanied bya measurement of target gene expression in said subject, and/or theproducts of the successful siRNA-targeting of mRNA. Other relevantcriteria can also be measured. The number and strength of doses areadjusted according to the subject's needs.

Example 27 Inhibition of BMPR1a in Humans

A human subject is treated with a siRNA targeted to a gene to inhibitexpression of the BMPR1a gene to treat a condition. In some instances,one or more additional siRNAs are co-administered, e.g., an siRNAtargeted to a HAMP, HFE2, HFE, TFR2, SMAD4, IL6R, BMP6, and/or NEO1gene.

A subject in need of treatment is selected or identified.

The identification of the subject can occur in a clinical setting, orelsewhere, e.g., in the subject's home through the subject's own use ofa self-testing kit.

At time zero, a suitable first dose of an siRNA is administered to thesubject. The siRNA is formulated as described herein. After a period oftime following the first dose, e.g., 7 days, 14 days, and 21 days, thesubject's condition is evaluated. This measurement can be accompanied bya measurement of target gene expression in said subject, and/or theproducts of the successful siRNA-targeting of mRNA. Other relevantcriteria can also be measured. The number and strength of doses areadjusted according to the subject's needs.

Example 28 Inhibition of SMAD4 in Humans

A human subject is treated with a siRNA targeted to a gene to inhibitexpression of the SMAD4 gene to treat a condition. In some instances,one or more additional siRNAs are co-administered, e.g., an siRNAtargeted to a HAMP, HFE2, HFE, TFR2, BMPR1a, IL6R, BMP6, and/or NEO1gene.

A subject in need of treatment is selected or identified.

The identification of the subject can occur in a clinical setting, orelsewhere, e.g., in the subject's home through the subject's own use ofa self-testing kit.

At time zero, a suitable first dose of an siRNA is administered to thesubject. The siRNA is formulated as described herein. After a period oftime following the first dose, e.g., 7 days, 14 days, and 21 days, thesubject's condition is evaluated. This measurement can be accompanied bya measurement of target gene expression in said subject, and/or theproducts of the successful siRNA-targeting of mRNA. Other relevantcriteria can also be measured. The number and strength of doses areadjusted according to the subject's needs.

Example 29 Inhibition of IL6R in Humans

A human subject is treated with a siRNA targeted to a gene to inhibitexpression of the IL6R gene to treat a condition. In some instances, oneor more additional siRNAs are co-administered, e.g., an siRNA targetedto a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, BMP6, and/or NEO1 gene.

A subject in need of treatment is selected or identified.

The identification of the subject can occur in a clinical setting, orelsewhere, e.g., in the subject's home through the subject's own use ofa self-testing kit.

At time zero, a suitable first dose of an siRNA is administered to thesubject. The siRNA is formulated as described herein. After a period oftime following the first dose, e.g., 7 days, 14 days, and 21 days, thesubject's condition is evaluated. This measurement can be accompanied bya measurement of target gene expression in said subject, and/or theproducts of the successful siRNA-targeting of mRNA. Other relevantcriteria can also be measured. The number and strength of doses areadjusted according to the subject's needs.

Example 30 Inhibition of BMP6 in Humans

A human subject is treated with a siRNA targeted to a gene to inhibitexpression of the BMP6 gene to treat a condition. In some instances, oneor more additional siRNAs are co-administered, e.g., an siRNA targetedto a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, and/or NEO1 gene.

A subject in need of treatment is selected or identified.

The identification of the subject can occur in a clinical setting, orelsewhere, e.g., in the subject's home through the subject's own use ofa self-testing kit.

At time zero, a suitable first dose of an siRNA is administered to thesubject. The siRNA is formulated as described herein. After a period oftime following the first dose, e.g., 7 days, 14 days, and 21 days, thesubject's condition is evaluated. This measurement can be accompanied bya measurement of target gene expression in said subject, and/or theproducts of the successful siRNA-targeting of mRNA. Other relevantcriteria can also be measured. The number and strength of doses areadjusted according to the subject's needs.

Example 31 Inhibition of NEO1 in Humans

A human subject is treated with a siRNA targeted to a gene to inhibitexpression of the NEO1 gene to treat a condition. In some instances, oneor more additional siRNAs are co-administered, e.g., an siRNA targetedto a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, and/or BMP6 gene.

A subject in need of treatment is selected or identified.

The identification of the subject can occur in a clinical setting, orelsewhere, e.g., in the subject's home through the subject's own use ofa self-testing kit.

At time zero, a suitable first dose of an siRNA is administered to thesubject. The siRNA is formulated as described herein. After a period oftime following the first dose, e.g., 7 days, 14 days, and 21 days, thesubject's condition is evaluated. This measurement can be accompanied bya measurement of target gene expression in said subject, and/or theproducts of the successful siRNA-targeting of mRNA. Other relevantcriteria can also be measured. The number and strength of doses areadjusted according to the subject's needs.

Tables

TABLE B SEQ ID NO DESCRIPTION SEQUENCE 1 Human HAMP -gactgtcactcggtcccagacaccagagcaagctcaagacccagcagtgggacagcc NM_021175.2agacagacggcacgatggcactgagctcccagatctgggccgcttgcctcctgctcctcctcctcctcgccagcctgaccagtggctctgttttcccacaacagacgggacaacttgcagagctgcaaccccaggacagagctggagccagggccagctggatgcccatgttccagaggcgaaggaggcgagacacccacttccccatctgcattttctgctgcggctgctgtcatcgatcaaagtgtgggatgtgctgcaagacgtagaacctacctgccctgcccccgtcccctcccttccttatttattcctgctgccccagaacataggtcttggaataaaatggctggttcttttgttttccaaaaaa 2 Cyno HAMP -tcaagacctagcagtgggacagccagacagacggcacgatggcactgagctcccaga EU076443.1tctgggccacttgcctcctcctccttctcctcctcgccagcctgaccagtggctccgttttcccacaacagacgggacaacttgcagagctgcaacctcaggacagagctggagccagggccagctggacgcccatgctccagaggcgaaggaggcgagacacccacttccccatctgcattttctgctgcggctgctgtcatcgatcaaagtgtgggatgtgctgcaggacgtagaaccttcctgccctgcccccatcccctcccttccttatttattcctgctgccccagaacacaggtcttggaataaaacggctgattcttttgttttcc 3 Mouse HAMP -agtccttagactgcacagcagaacagaaggcatgatggcactcagcactcggaccca NM_032541.1ggctgcctgtctcctgcttctcctccttgccagcctgagcagcaccacctatctccatcaacagatgagacagactacagagctgcagcctttgcacggggaagaaagcagggcagacattgcgataccaatgcagaagagaaggaagagagacaccaacttccccatctgcatcttctgctgtaaatgctgtaacaattcccagtgtggtatctgttgcaaaacatagcctagagccacatcctgacctctctacacccctgcagcccctcaaccccattatttattcctgccctccccaccaatgaccttgaaataaagacgattttattttcaaaaaaa aaaaaaaaaaa 4Rat HAMP - cacgagggcaggacagaaggcaagatggcactaagcactcggatccaggctgcctgtNM_053469.1 ctcctgcttctcctcctggccagcctgagcagcggtgcctatctccggcaacagacgagacagactacggctctgcagccttggcatggggcagaaagcaagactgatgacagtgcgctgctgatgctgaagcgaaggaagcgagacaccaacttccccatatgcctcttctgctgtaaatgctgtaagaattcctcctgtggtctctgttgcataacatagagagccaagagccttgtcctgacctctcaacacactgcctcccctccgccccattatttattcctgtcctaccccagcaatgaccttg 5 Human HFE2 -accgtcaactcagtagccacctccctccctgctcagctgtccagtactctggccagc NM_213652.3catatactcccccttccccccataccaaaccttctctggttccctgacctcagtgagacagcagccggcctggggacctgggggagacacggaggaccccctggctggagctgacccacagagtagggaatcatggctggagaattggatagcagagtaatgtttgacctctggaaacactcaccatcatatttaagaacatgcaggaatgcattgatcagaaggtgtatcaggctgaggtggataatcttcctgtagcctttgaagatggttctatcaatggaggtgaccgacctgggggatccagtttgtcgattcaaactgctaaccctgggaaccatgtggagatccaagctgcctacattggcacaactataatcattcggcagacagctgggcagctctccttctccatcaaggtagcagaggatgtggccatggccttctcagctgaacaggacctgcagctctgtgttggggggtgccctccaagtcagcgactctctcgatcagagcgcaatcgtcggggagctataaccattgatactgccagacggctgtgcaaggaagggcttccagtggaagatgcttacttccattcctgtgtctttgatgttttaatttctggtgatcccaactttaccgtggcagctcaggcagcactggaggatgcccgagccttcctgccagacttagagaagctgcatctcttcccctcagatgctggggttcctctttcctcagcaaccctcttagctccactcctttctgggctctttgttctgtggctttgcattcagtaaggggaccatcagtcccattactagtttggaaatgatttggagatacagattggcatagaagaatgtaaagaatcattaaaggaagcagggcctaggagacacgtgaaacaatgacattatccagagtcagatgaggctgcagtccagggttgaaattatcacagaataaggattctgggcaaggttactgcattccggatctctgtggggctcttcaccaatttttccagcctcatttatagtaaacaaattgttctaatccatttactgcagatttcacccttataagtttagaggtcatgaaggttttaatgatcagtaaagatttaagggttgagatttttaagaggcaagagctgaaagcagaagacatgatcattagccataagaaactcaaaggaggaagacataattagggaaagaagtctatttgatgaatatgtgtgtgtaaggtatgttctgctttcttgattcaaaaatgaagcaggcattgtctagctcttaggtgaagggagtctctgcttttgaagaatggcacaggtaggacagaagtatcatccctaccccctaactaatctgttattaaagctacaaattcttcacaccatcaaaaaaaaaaaaa aaaaaa 6Rhesus HFE2 - cttctctggctccctgacctcagtgagacagcagccggcctggggacctgggggagaXM_001092987.1 catggagaaagagacggaggaccccctggctggagctgacccacagagtagggaatcatggctggagaattggatagcagagtaatgtttgacctctggaaacaccaaatttcttttttcagtcacttacagggcttccggtcaaaattcactaggtaggagggtcatcagctgggaagaaccggcgcctggggaacctggctggataggtatgggggagcaaggccagtcccctagtcccaggtcctcccatggcagtcccccaactctaagcactctcactctcctgctgctcctctgtggacatgctcattctcaatgcaagatcctccgctgcaatgctgagtatgtatcgtccactctgagccttagaggtggcggttcatcaggagcacttcgaggaggaggaggaggaggaggccggggtggaggggtgggctctggcggcctctgtcgagccctccgctcctatgcgctctgcactcggcgcaccgcccgcacctgccgtggggacctcgccttccattcggcggtacatggcatcgaagacctgatgatccagcacaactgctcgcgccagggccctacagcccctcccccgccccggggccccgcccttccaggcgcaggctccggcctccctgccccggacccttgtgactatgaaggccggttttcccggctgcatggtcgtcccccggggttcttgcattgcgcttccttcggggacccccatgtgcgcagcttccaccaccattttcacacatgccgtgtccaaggagcttggcctctactggataacgacttcctctttgtccaagccaccagctcccccatggcgttgggggccaacgctaccgctacccggaagctcaccatcatatttaagaacatgcaggaatgcattgatcagaaggtctatcaggctgaggtggataatcttcctgcagcctttgaagatggttctgtcaatggaggtgaccgacctgggggatccagtttgtcgattcaaactgctaaccctgggaaccacgtggagatccaagctgcctacattggcacaactataatcattcggcagacagctgggcagctctccttctccatcaaggtagcagaggatgtggccatggccttctcagctgaacaggacctgcagctctgtgttggggggtgccctccaagtcagcgactctctcgatcagagcgcagtcgtccgggagctataaccattgatactgccagacggctgtgtaaggaagggcttccagtggaagatgcttacttccattcctgtgtctttgatgttttaatttctggtgatcccaactttactgtggcagctcaggcagcactggaggatgcccgagccttcctgccagacttagataagctgcatctcttcccttcagatgctggggtttctctttcctcagcaaccttcctagccccactcctttctgggctctttgttctgtggctttgcattcagtaaggaagccatcagtcctattactagtttggaaatgatttggggatagagattggcatagaagaatgtaaacaatcattaaaggaagcagggcccagaagacacatgaaacaatgacatcatccagagtcagatgaggctgcagtccagggttgaaatgatcacagaataaggattctgggcaaggtttctgcattccagacctcttcgccaaattttccagccccatttacagtaaacaaattgttctttccatttactgcagatttcaccctataagcttagaggtcatgaaggttttaacaatcagtaaagacttaagggttgagatttttaagaggcaagagctgaaagcagaagacatgatcattagccataagaaactcaaaggaagaagaaataattagggaaagaagtctatttgatgaatatgtgtgtgtaaggtatgttctgctttcttggttcaaaaatgaagcgggcgttgtctagctcttaggtgaagggagtctctgctttggaagaacggcacaggtaggacagaagtatcatccctacccctaactgatctgttattaaagctacaaattcttcacaccgtc 7 Mouse HFE2 -ggctctctgacctgagtgagactgcagccattccggggcaatcatggagaaagagat NM_027126.4gggggaccccctggctggagcagaccaacagaataggcaactatggctcgagaacccagtatcagagtaatgcttgacctcgggaaacatcacagaagtacccagagaaattcactaggtaggaggctcatcatctgggaagaaccggtgcctggggggacctggctggataggtatgggccagtcccctagtccccggtccccccacggcagccctccaactctaagcaccctcactctcctgctgctcctctgtggacaggctcactcccagtgcaagatcctccgctgcaatgccgagtatgtctcgtccactctgagtcttcggggaggtggctcaccggacacgccgcgtggaggcggccgtggtgggctggcctcaggtggcttgtgtcgcgccctgcgctcctacgctctctgcacgcggcgcacggcccgcacctgccgcggggaccttgctttccactctgcggtgcatggcatagaggacctgatgatccagcacaactgctcacgccagggtcccacggccccgcccccggcccggggccccgccctgcccggggccgggccagcgcccctgaccccagatccctgtgactatgaggcccggttttccaggctgcacggtcgagccccgggcttcttgcattgcgcatcctttggagatccccatgtgcgcagtttccacaaccaatttcacacatgccgtgtccaaggagcttggcccttgctagataacgacttcctctttgtccaggccaccagctccccggtttcgtcgggagccaacgctaccaccatccggaagatcactatcatatttaaaaacatgcaggaatgcattgaccagaaagtctaccaggctgaggtggacaatcttcctgcagcctttgaagatggttctatcaatgggggcgaccgacctgggggctcgagtttgtccattcaaactgctaaccttgggagtcacgtggagattcgagctgcctacattggaacaactatcatcattcgacagacagctgggcagctctccttctccatcagggtagcagaggatgtggcgcgggccttctccgcagagcaggacctacagctgtgtgttgggggatgccctccgagccagcgactctctcgctcagagcgcaaccgccgtggggctatagccatagatactgccagaaggctgtgtaaggaagggcttccggttgaagatgcctacttccaatcctgcgtctttgatgtttcagtctccggtgaccccaactttactgtggcagctcagacagctctggacgatgcccgaatcttcttgacggatttagagaacttacatctctttccctcagatgcggggcctcccctctctcctgccatctgcctagtcccgcttctttcggccctctttgttctgtggctttgcttcagtaagtaggccagcaacccatgactggtttggaaacgatttgaggatagaggttggtgtgagaaaccacaaagatgtgccaaaggaaacagcggggacaggagacaacacttactcaatcagatgaggttgcagtccagggctgaaatgaccctagaataaagattctgggccagggttttgcactccagaccttggtgtgggctattcaccatggatttcccagttagtgatttcccacttgtaatgaaattccactctccatacacctgataccactcctacaagcctagagattgtgagagtgctaatgaccagtgaaacattaaaggactgagatatcgtaaaggcaaaaacatgattctctttgagaaagtcaaaagaggagaagctaattaggaaaagcttttggttcagaaacgaagtgggcattgtctggcagaggaagtcagcttttggagactggcaccaactcagaaacgggcatttccatcccttcctaatctgttattaaagcgattagttctccatcctg 8 Rat HFE2 -cggggacagacatggagaaggagatggaggaccccctggctggagcagaccaacaga NM_001012080.1ataggcaactatggctggagaaccgggtatcagagtaatgcttgacctcgggaaacaccaaatttcttcttccgatcgcagaagtagtactcggcgaaattcactaggtaggaggctcctcatctgggaagaaccggtgcctggggggacctggctggataggtatgggggatcgaggccggtcccctagtctccggtccccccatggcagtcctccaactctaagcaccctcactctcctgctgctcctctgtggacaggctcactcccagtgcaagatcctccgctgcaatgccgagtacgtctcgtccactctgagccttcggggagggggctcaccggacacgccacatggaggcggccgtggtgggccggcctcaggtggcttgtgtcgcgccctgcgctcctacgctctctgcacgcggcgcaccgcccgcacctgccgcggggacctcgctttccactccgcggtgcatggcatagaggacctgatgatccagcacaactgctcacgccagggtcccacggcctcgcccccggcccggggtcctgccctgcccggggccggcccagcgcccctgaccccagatccctgtgactatgaagcccggttttccaggctgcacggtcgaaccccgggtttcttgcattgtgcttcctttggagacccccatgtgcgcagcttccacaatcactttcacacatgccgcgtccaaggagcttggcccctactagataacgacttcctctttgtccaagccaccagctccccggtagcatcgggagccaacgctaccaccatccggaagatcactatcatatttaaaaacatgcaggaatgcattgaccagaaagtctaccaggctgaggtagacaatcttcctgcagcctttgaagatggttctgtcaatgggggcgaccgacctgggggctcgagtttgtccattcaaactgctaaccttgggagccacgtggagattcgagctgcctacattggaacaactataatcgttcgtcagacagctggacagctctccttctccatcagggtagcggaggatgtggcacgggccttctctgctgagcaggatctacagctgtgtgttgggggatgccctccgagccagcgactctctcgctcagagcgcaatcgccgtggggcgatagccatagatactgccagaaggttgtgtaaggaagggcttccggttgaagatgcctacttccaatcctgcgtctttgatgtttcagtctccggtgaccccaactttactgtggcagctcagtcagctctggacgatgcccgagtcttcttgaccgatttggagaacttgcaccttttcccagtagatgcggggcctcccctctctccagccacctgcctagtccggcttctttcggtcctctttgttctgtggttttgcattcagtaagtaggccagcaacccgtgactagtttggaaacggtttgaggagagaggttgatgtgagaaaacacaaagatgtgccaaaggaaacagtggggacaggagacaacgaccttactcaatcacacgaggttgcagtccagggctgaaatgaccctagaataaagattctgagacagggttttgcactccagaccttggtatgggctccccatgaatttccccattagtgatttcccacttgtagtgaaattctactctctgtacacctgatatcactcctgcaaggctagagattgtgagagcgctaagggccagcaaaacattaaagggctgagatatcttaaaggcagaaactagaaaaggggaaaccatgattatctataagaaaatcaaaagaggggtttgggaatttagctcagtggtagagcacttgcctagcaagcgcaaggccctgggttcggtccccagctcctaaaaaagaaaaaaaaaatcaaaagagaaaaaactaattaaggcaagctttttggttcagaaatgaagtgggcattgtctggcagaggaagtcagcttttggagactggcaccaacatctccacccttcctactctgttattaaagtgacgaattccccaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagg 9 Human TFR2 -cgctgggggacagcctgcaggcttcaggaggggacacaagcatggagcggctttggg NM_003227.3gtctattccagagagcgcaacaactgtccccaagatcctctcagaccgtctaccagcgtgtggaaggcccccggaaagggcacctggaggaggaagaggaagacggggaggagggggcggagacattggcccacttctgccccatggagctgaggggccctgagcccctgggctctagacccaggcagccaaacctcattccctgggcggcagcaggacggagggctgccccctacctggtcctgacggccctgctgatcttcactggggccttcctactgggctacgtcgccttccgagggtcctgccaggcgtgcggagactctgtgttggtggtcagtgaggatgtcaactatgagcctgacctggatttccaccagggcagactctactggagcgacctccaggccatgttcctgcagttcctgggggaggggcgcctggaggacaccatcaggcaaaccagccttcgggaacgggtggcaggctcggccgggatggccgctctgactcaggacattcgcgcggcgctctcccgccagaagctggaccacgtgtggaccgacacgcactacgtggggctgcaattcccggatccggctcaccccaacaccctgcactgggtcgatgaggccgggaaggtcggagagcagctgccgctggaggaccctgacgtctactgcccctacagcgccatcggcaacgtcacgggagagctggtgtacgcccactacgggcggcccgaagacctgcaggacctgcgggccaggggcgtggatccagtgggccgcctgctgctggtgcgcgtgggggtgatcagcttcgcccagaaggtgaccaatgctcaggacttcggggctcaaggagtgctcatatacccagagccagcggacttctcccaggacccacccaagccaagcctgtccagccagcaggcagtgtatggacatgtgcacctgggaactggagacccctacacacctggcttcccttccttcaatcaaacccagttccctccagttgcatcatcaggccttcccagcatcccagcccagcccatcagtgcagacattgcctcccgcctgctgaggaagctcaaaggccctgtggccccccaagaatggcaggggagcctcctaggctccccttatcacctgggccccgggccacgactgcggctagtggtcaacaatcacaggacctccacccccatcaacaacatcttcggctgcatcgaaggccgctcagagccagatcactacgttgtcatcggggcccagagggatgcatggggcccaggagcagctaaatccgctgtggggacggctatactcctggagctggtgcggaccttttcctccatggtgagcaacggcttccggccccgcagaagtctcctcttcatcagctgggacggtggtgactttggaagcgtgggctccacggagtggctagagggctacctcagcgtgctgcacctcaaagccgtagtgtacgtgagcctggacaacgcagtgctgggggatgacaagtttcatgccaagaccagcccccttctgacaagtctcattgagagtgtcctgaagcaggtggattctcccaaccacagtgggcagactctctatgaacaggtggtgttcaccaatcccagctgggatgctgaggtgatccggcccctacccatggacagcagtgcctattccttcacggcctttgtgggagtccctgccgtcgagttctcctttatggaggacgaccaggcctacccattcctgcacacaaaggaggacacttatgagaacctgcataaggtgctgcaaggccgcctgcccgccgtggcccaggccgtggcccagctcgcagggcagctcctcatccggctcagccacgatcgcctgctgcccctcgacttcggccgctacggggacgtcgtcctcaggcacatcgggaacctcaacgagttctctggggacctcaaggcccgcgggctgaccctgcagtgggtgtactcggcgcggggggactacatccgggcggcggaaaagctgcggcaggagatctacagctcggaggagagagacgagcgactgacacgcatgtacaacgtgcgcataatgcgggtggagttctacttcctttcccagtacgtgtcgccagccgactccccgttccgccacatcttcatgggccgtggagaccacacgctgggcgccctgctggaccacctgcggctgctgcgctccaacagctccgggacccccggggccacctcctccactggcttccaggagagccgtttccggcgtcagctagccctgctcacctggacgctgcaaggggcagccaatgcgcttagcggggatgtctggaacattgataacaacttctgaggccctggggatcctcacatccccgtcccccagtcaagagctcctctgctcctcgcttgaatgattcagggtcagggaggtggctcagagtccacctctcattgctgatcaatttctcattacccctacacatctctccacggagcccagaccccagcacagatatccacacaccccagccctgcagtgtagctgaccctaatgtgacggtcatactgtcggttaatcagagagtagcatcccttcaatcacagccccttcccctttctggggtcctccatacctagagaccactctgggaggtttgctaggccctgggacctggccagctctgttagtgggagagatcgctggcaccatagccttatggccaacaggtggtctgtggtgaaaggggcgtggagtttcaatatcaataaaccacctgatatcaataagccaaaa 10 Human TFR2 -ccctgcccctggcgaccccacgtctctggcatccttccctcttccctccctctcctc NM_001206855.1cgggcgcccagaaaagtccccacctctccccgcttaggcaaaccagccttcgggaacgggtggcaggctcggccgggatggccgctctgactcaggacattcgcgcggcgctctcccgccagaagctggaccacgtgtggaccgacacgcactacgtggggctgcaattcccggatccggctcaccccaacaccctgcactgggtcgatgaggccgggaaggtcggagagcagctgccgctggaggaccctgacgtctactgcccctacagcgccatcggcaacgtcacgggagagctggtgtacgcccactacgggcggcccgaagacctgcaggacctgcgggccaggggcgtggatccagtgggccgcctgctgctggtgcgcgtgggggtgatcagcttcgcccagaaggtgaccaatgctcaggacttcggggctcaaggagtgctcatatacccagagccagcggacttctcccaggacccacccaagccaagcctgtccagccagcaggcagtgtatggacatgtgcacctgggaactggagacccctacacacctggcttcccttccttcaatcaaacccagttccctccagttgcatcatcaggccttcccagcatcccagcccagcccatcagtgcagacattgcctcccgcctgctgaggaagctcaaaggccctgtggccccccaagaatggcaggggagcctcctaggctccccttatcacctgggccccgggccacgactgcggctagtggtcaacaatcacaggacctccacccccatcaacaacatcttcggctgcatcgaaggccgctcagagccagatcactacgttgtcatcggggcccagagggatgcatggggcccaggagcagctaaatccgctgtggggacggctatactcctggagctggtgcggaccttttcctccatggtgagcaacggcttccggccccgcagaagtctcctcttcatcagctgggacggtggtgactttggaagcgtgggctccacggagtggctagaaggctacctcagcgtgctgcacctcaaagccgtagtgtacgtgagcctggacaacgcagtgctgggggatgacaagtttcatgccaagaccagcccccttctgacaagtctcattgagagtgtcctgaagcaggtggattctcccaaccacagtgggcagactctctatgaacaggtggtgttcaccaatcccagctgggatgctgaggtgatccggcccctacccatggacagcagtgcctattccttcacggcctttgtgggagtccctgccgtcgagttctcctttatggaggacgaccaggcctacccattcctgcacacaaaggaggacacttatgagaacctgcataaggtgctgcaaggccgcctgcccgccgtggcccaggccgtggcccagctcgcagggcagctcctcatccggctcagccacgatcgcctgctgcccctcgacttcggccgctacggggacgtcgtcctcaggcacatcgggaacctcaacgagttctctggggacctcaaggcccgcgggctgaccctgcagtgggtgtactcggcgcggggggactacatccgggcggcggaaaagctgcggcaggagatctacagctcggaggagagagacgagcgactgacacgcatgtacaacgtgcgcataatgcgggtggagttctacttcctttcccagtacgtgtcgccagccgactccccgttccgccacatcttcatgggccgtggagaccacacgctgggcgccctgctggaccacctgcggctgctgcgctccaacagctccgggacccccggggccacctcctccactggcttccaggagagccgtttccggcgtcagctagccctgctcacctggacgctgcaaggggcagccaatgcgcttagcggggatgtctggaacattgataacaacttctgaggccctggggatcctcacatccccgtcccccagtcaagagctcctctgctcctcgcttgaatgattcagggtcagggaggtggctcagagtccacctctcattgctgatcaatttctcattacccctacacatctctccacggagcccagaccccagcacagatatccacacaccccagccctgcagtgtagctgaccctaatgtgacggtcatactgtcggttaatcagagagtagcatcccttcaatcacagccccttcccctttctggggtcctccatacctagagaccactctgggaggtttgctaggccctgggacctggccagctctgttagtgggagagatcgctggcaccatagccttatggccaacaggtggtctgtggtgaaaggggcgtggagtttcaatatcaataaaccacctgatatcaataagccaaaa 11 Rhesus TFR2 -accccaggacctgcgctcagggagcaggcaggtgtggggctgtggagagattggcag XM_001113151.2gggagagcacagccgcttgtgctctggcctggactcaggggccacgtctggaaggttggaccgaggccaggactgtgcccccacccttgggggtggtaaggagcagccttggctcaggctttctgccagggctgataaggagccctcctggggctcccacaaacggtttatcggtttatcactggggacagcctgcaggcttcaggagggggcacaagcatggagcagctttggggtctactccagagagcgcaacaactgtccccaagatcctctcagaccgtctaccagcgtgtggaaggcccccagaaagggcacctggaggaggaagaggaagacggggaggagacactggcccacttctgccccatggagctgaagggccctgagcccctgggctctagacccaggcagccaaacctcattccctgggcagcagcaggacggagggctgccccctacctggtcctgactgctctactgatcttcactggggccttccttctgggctacgtcgccttccgagggtcctgccagacatgcggagactccgtgttggtggtcagtgaggacgtcaactatgagcctgacctggatttccaccggggcacactgtactggagcgacctccaggccatgttcctgcagttcctgggggaggggcgcctggaggacaccatcaggcaaaccagccttcgggaacgggtggcaggctcggccgggatggccgctctgactcaggatatccgcgcggcgctctctcgccagaagctggaccacgtgtggaccgacacgcactacgtggggctgcaattcccggacccggctcaccccaacaccctgcactgggtcgatgaggccgggaaggtcggagagcagctgccgctagaggaccctgacgtctactgcccctacagcgccatcggcaacgtcacgggagagctggtgtacgcccactacgggcggcccgaagacctgcaggacctgcgggccaggggcgtggacccagcgggccgcctgctgctagtgcgcgtgggggtgatcagcttcgcccagaaggtgaccaatgctcaggactttggggctcaaggagtgctcatatacccagagccagcggacttctcccaggacccacacaagccaagcctgtccagccagcaggctgtgtatggacatgtgcacctgggaactggagacccctacacgcctggcttcccttccttcaatcaaacccagttccctccagttgcatcatcgggccttcccagcatcccagcccagcccatcactgcagacattgcctcccgcctgctgaggaagctcaaaggccctgtggccccccaggaatggcaggggagcctcctaggctccccttatcacctgggccccgggccacgactgcggctagcggtcaacaaccacaggacctccacccccatcaacaacatctttggctgcatcgaaggccgctcagagccagatcactatgttgtcatcggggcccagagggatgcgtggggcccaggagcagctaaatccgctgtggggacagctatactcctggagctggtgcggaccttttcctccatggtgagcaacggcttccggccccgcagaagtctcctcttcatcagctgggatggcggtgactttgggagcgtgggctccacagagtggctagagggctacctcagtgtgctgcacctcaaagctgtagtgtacgtgagcctggacaacgcagtgctgggggatgacaagtttcatgccaagaccagcccccttctgacaagtctcattgagagtgtcctgaaacaggcaagagcaccccaggaatggctgaccctgcagtgggtgtactccgcgcggggggactacatccgggcggcggagaagctgcggcaggagatctacagctcggaggagagagacgagcgactgacacgcatgtacaacgtgcgcataatgcgggtggagttctacttcctttcccagtacgtgtcgccggccgactccccgttccgccacatcttcatgggccgcggagaccacacgctgggcgccctgctggaccacctgcggctgctgcgctccaacagctccgggacccccggggccacctcctccgccgtcttccaggagagtcgcttccggcgtcagctagccctgctcacctggacgctgcaaggggcagccaatgcgcttagcggggacgtctggaacattgataacaacttctgagaccctggggatcctcagatccccctgtccccttgtcgagagctcctctgctcctcgcttcaatgattcagggtcagggaggtggctcagagtccacctctcattgctgatcgacttctcattacccctacacgtctctccacggagcccagactgcagcacagatatccacacaccccagccctgcagtgtagctgactctaatgtgatggtcatactgtcggttaatcagagagcagtatcccttcaatcacaaccccttcccctttctggggtcctccatacctagagactaggccttgggacctggccagctctcttagcgggagagatcgctggcaccatagccttatggccaacaggtggtctgtggtgaaaggggcatggagtttcaatgtc 12 Mouse TFR2 -gagcatggtccaagaaacccagagacctgttgctgagctgaacttggctgctgtgtc NM_015799.3ttcccactcaggactcggctttgacagctgcaggtcctggtgtcttcgtcgcggcttggatttcaaactggaggagttcaggagggggcacaagcatggagcaacgttggggtctacttcggagagtgcaacagtggtccccaagaccctctcagaccatctacagacgcgtggaaggccctcagctggagcacctggaggaggaagacagggaggaaggggcggagcttcctgcccagttctgccccatggaactcaaaggccctgagcacttaggctcctgtcccgggaggtcaattcccataccctgggctgcagcaggtcgaaaggctgccccctatctggtcctgatcaccctgctaatcttcactggggccttcctcctaggctacgtggcctttcgagggtcctgccaggcgtgtggggactccgtgttggtggtcgatgaagatgtcaaccctgaggactccggccggaccacgttgtactggagcgacctccaggccatgtttctccggttccttggggaggggcgcatggaagacaccatcaggctgaccagcctccgggaacgcgtggctggctcagccagaatggccaccctggtccaagatatcctcgataagctctcgcgccagaagctggaccacgtgtggactgacacgcactacgtgggacttcagttcccagatccggctcacgctaacaccctgcactgggtggatgcagacgggagcgtccaggagcagctaccgctggaggatccggaagtctactgtccctacagcgccaccggcaacgccacgggcaagctggtgtacgcccactacgggcggtcggaggacctacaggacctaaaagccaagggcgtggagctggccggcagcctcctgctagtgcgagttggaattactagcttcgcccagaaggtagccgttgcccaggactttggggctcaaggagtgctgatataccctgacccatcagacttctcccaggatccccacaagccaggcctgtctagccaccaggctgtgtacggacatgtgcacctgggaactggagacccttacacacctggcttcccgtccttcaatcaaacccagttccctccagtagaatcatcaggccttcccagcatccccgcccagcccatcagtgctgacattgctgatcaattgctcaggaaactcacaggccccgtggctccccaggagtggaaaggtcacctctcaggctctccttatcggctgggacctgggcccgacttacgccttgtggtcaacaaccacagagtctctacccccatcagtaacatctttgcgtgcatcgagggctttgcagagccagatcactatgttgtcattggggcccagagggatgcatggggcccaggagcagccaagtctgcagtggggactgccatcctgctggagctggttcggaccttctcttccatggtcagcaatgggttcagacctcgaagaagtcttttgttcatcagctgggacggaggtgactttggcagcgtgggagccacagagtggttggagggctacctcagcgtgctacacctcaaagctgttgtgtacgtgagcctggacaactccgtgttgggagatggcaaattccatgctaagaccagcccccttctcgtcagcctcattgagaatatcttgaagcaggtggactcccctaaccatagtggacagaccctctatgaacaagtggcactcacccaccccagctgggatgctgaagtgattcagcccctgcccatggacagcagtgcatattccttcacagcctttgcgggggtcccagctgtggagttctccttcatggaggatgatcgggtgtacccattcctgcacacgaaggaggacacatatgagaatctgcacaagatgctgcgaggtcgcctgcccgccgtggtccaggcagtggctcagctcgcgggccagctcctcatccgactgagccacgatcacctactgccgctagacttcggccgctatggagacgtggttctcaggcacatcggcaacctcaatgagttctctggggacctcaaggagcgcgggctgaccctgcagtgggtgtactctgcaaggggggactacatccgtgcggcggaaaagctgcggaaggagatttacagctcggagcggaacgatgagcgtctgatgcgcatgtacaacgtgcgcatcatgagggtggagttctacttcctgtcccagtatgtgtcgccagccgactccccattccgccacattttcctaggccaaggcgaccacactttgggtgccctggtagaccacctgcggatgctgcgcgccgatggctcaggagccgcctcttcccggttgacagcaggtctgggcttccaggagagtcgcttccggcgccagctggcgctgctcacctggacactgcagggggcagccaacgctctcagtggcgacgtttggaacattgacaataacttttgaagccaaaagccctccatgggccccacgtgattctcctttctccctctttgagtggtgcaggcaaaggaggtgcctgagattgtaacctattcttaacacccttggtcctgcaatgctggtgcgccatattttctcagtgtggttgtcatgccgttgcttacccagaaagcggttttcttcccatcacaggcccttctgtcttcaggagcaaagttccccatatctagagactatctagatgctgggatctgatcagctctcttagagagtgagatggacagcgtcattattttatgacacatgagctacggtatgtgagcagcccaaggggattagatgtcaataaaccaattgtaacccctgttgtccatacgcaa 13 Rat TFR2 -aaatccagagacctgttgctgagttgaacttggctgctgtgtcttcccactcaggac NM_001105916.1tcggctttgacaggcacgaggcagggactggggtgagcccctacctctcagatctttctggacctggctgcgggtcctgggatcttcagcgcggcttggatttcaaactggaggggttcaggagggggcacaagcatggaacaacgttggggtctacttcggaaagtgcaacagtggtccccaagaccctctcagaccatctacagacgtgtggaaggccctcaactggagaacctagaggaggaagatagggaggaaggggaggagcttcctgcccagttctgccccatggaactcaaaggccctgagcgcttaggctcctgtcctgggaggtccattcccataccctgggctgcagcaggtcgaaaggctgctccctatctggtcctgaccaccctgctaatcttcactggggccttcctcctgggctacgtggcctttcgagggtcctgccaggcatgtggggactctgtgttggtggttggtgaagatgtcaactctgaggactccagccggggcacgttgtactggagtgacctccaggacatgtttctccggttccttggggagggacgcatggaggacaccatcaggctgaccagcctccgggaacgcgtggccggctcagccagaatggccaccctggtccaagacatcctcgataagctctcgcgccagaagctggaccacgtgtggactgacacgcactatgtgggacttcagttcccggacccggctcaccctaacaccctgcactgggtgggtgcagacgggagcgtccaagagcagctaccgctggaggatccggaagtctactgtccctacagcgccacgggcaacgccacgggcaagcttgtgtacgcccactacgggcggcgggaggacctgcaggacctgaaagccaaggacgtggagctggccggcagcctcctgctagtgcgtgctgggattacaagcttcgcccagaaggtagccattgcccaggactttggggcccacggagtgctgatataccctgacccagcggacttctcccaagacccccacaagccaggcctgtctagtgacagggctgtgtatggacatgtgcacctgggaactggggacccttacacgcctggcttcccgtccttcaatcaaacccagttccctccagtagaatcatcggggcttcccaacatccctgcccagcccatcagtgccgacgttgctgatcgcttgctcaggaaactcacaggtcccgtggctcctcaggaatggaagggtcgcctctcagactctccgtatcgcctgggacctgggccaggcttacgccttgtggtcaacaaccacagaacctctactcccatcagtaacatctttgcgtgcatcgagggcttcgcagagccagatcactatgtcgttatcggggcccagagggatgcctggggcccaggagcagccaagtctgcagtggggactgccatcctcctggagctggttcggaccttttcctccatggtcagcagtggctttagacctcgaagaagtcttttgttcatcagctgggacggaggtgactttggcagcgtgggagccacggagtggttggagggctacctcagcgtgctacacctcaaagctgtcgtgtatgtgagcctggacaactccgtgttgggagacggcaaattccatgctaagaccagcccccttctcgtcagcctcattgagaatatcctgaagcaggtggattcccctaaccacagtggacagacactctacgatcaagtggcattcacccacccaagctgggatgctgaagtgatccagcccctgcccatggacagcagcgcatattccttcacagcttttgcgggcgtcccagctgtggagttctccttcatggaggacgatagggtgtacccattcctgcacacgaaggaggacacgtatgagaatctgcacaagatgctgcgaggtcgcctgcccgccgtggtcctagcagtggctcagctcgctggtcagctcctcatccgactgagccacgatcacctactgccgctggacttcggccgctacggagacgtggtcctcaggcacatcggcaaccttaatgagttctctggggacctcaaggcgcgcgggctgaccctgcagtgggtgtactctgcaaggggggactatatccgggcggcggagaagctgcggaaggagatttacagctcggagcagagcgatgagcgtctgatgcgcatgtacaacgtgcgcatcatgagggtggagttctacttcctgtcccagtacgtgtcgccggccgactccccattccgccacattttcctaggccaaggcgaccacactttgggtgccctggtggaacacctacggatgctgcgctccgatggctcaggagctgcctcttctgggttgagcccaggtctgggcttccaggagagtcgcttccggcgacagctggcgctgctcacgtggacgctacagggggcagccaacgcactcagtggcgacgtttggaacatcgacaataacttttgaggccagaagtcctccatgggccccacgtgattctcctttctccctatttgagtggtgcaggcaacggaggtgcctgagagcaacctatcctcattaacaaccttggtcctgcaacgccagtgagacatattttctcagtgtgactgttataccactgtttatccagaaagcggttttcttcccatcactggcctctctgccttcaggagcatagttccccatatctagaaaccatctagacactgggatccagctctcttagcgggtgagatggatagcgtcattttcttatgacacacaagtggtatgtgggtggcccaagggggattagatgtcaataaaccatttacctggtaacctctgttgtccataagc 14 Human HFE -ctaaagttctgaaagacctgttgcttttcaccaggaagttttactgggcatctcctg NM_139006.2agcctaggcaatagctgtagggtgacttctggagccatccccgtttccccgccccccaaaagaagcggagatttaacggggacgtgcggccagagctggggaaatgggcccgcgagccaggccggcgcttctcctcctgatgcttttgcagaccgcggtcctgcaggggcgcttgctgcgttcacactctctgcactacctcttcatgggtgcctcagagcaggaccttggtctttccttgtttgaagctttgggctacgtggatgaccagctgttcgtgttctatgatcatgagagtcgccgtgtggagccccgaactccatgggtttccagtagaatttcaagccagatgtggctgcagctgagtcagagtctgaaagggtgggatcacatgttcactgttgacttctggactattatggaaaatcacaaccacagcaaggagtcccacaccctgcaggtcatcctgggctgtgaaatgcaagaagacaacagtaccgagggctactggaagtacgggtatgatgggcaggaccaccttgaattctgccctgacacactggattggagagcagcagaacccagggcctggcccaccaagctggagtgggaaaggcacaagattcgggccaggcagaacagggcctacctggagagggactgccctgcacagctgcagcagttgctggagctggggagaggtgttttggaccaacaagtgaccactctacggtgtcgggccttgaactactacccccagaacatcaccatgaagtggctgaaggataagcagccaatggatgccaaggagttcgaacctaaagacgtattgcccaatggggatgggacctaccagggctggataaccttggctgtaccccctggggaagagcagagatatacgtgccaggtggagcacccaggcctggatcagcccctcattgtgatctgggagccctcaccgtctggcaccctagtcattggagtcatcagtggaattgctgtttttgtcgtcatcttgttcattggaattttgttcataatattaaggaagaggcagggttcaagaggagccatggggcactacgtcttagctgaacgtgagtgacacgcagcctgcagactcactgtgggaaggagacaaaactagagactcaaagagggagtgcatttatgagctcttcatgtttcaggagagagttgaacctaaacatagaaattgcctgacgaactccttgattttagccttctctgttcatttcctcaaaaagatttccccatttaggtttctgagttcctgcatgccggtgatccctagctgtgacctctcccctggaactgtctctcatgaacctcaagctgcatctagaggcttccttcatttcctccgtcacctcagagacatacacctatgtcatttcatttcctatttttggaagaggactccttaaatttgggggacttacatgattcattttaacatctgagaaaagctttgaaccctgggacgtggctagtcataaccttaccagatttttacacatgtatctatgcattttctggacccgttcaacttttcctttgaatcctctctctgtgttacccagtaactcatctgtcaccaagccttggggattcttccatctgattgtgatgtgagttgcacagctatgaaggctgtacactgcacgaatggaagaggcacctgtcccagaaaaagcatcatggctatctgtgggtagtatgatgggtgtttttagcaggtaggaggcaaatatcttgaaaggggttgtgaagaggtgttttttctaattggcatgaaggtgtcatacagatttgcaaagtttaatggtgccttcatttgggatgctactctagtattccagacctgaagaatcacaataattttctacctggtctctccttgttctgataatgaaaattatgataaggatgataaaagcacttacttcgtgtccgactcttctgagcacctacttacatgcattactgcatgcacttcttacaataattctatgagataggtactattatccccatttcttttttaaatgaagaaagtgaagtaggccgggcacggtggctcac gcctgtaatcccag15 Rhesus HFE -ttttactgggcatctcctgagcctaggcaatagctgtagggtgacttctggagccat XM_001085598.2cgccgtttccccgccccaccaaagaagcggagacttaaaggggacgtgcagtcagagctggggaaatgggcccgcgagccaggccggcgcttctcctcctgatgcttttgcagaccgcggtcctgcaggggcgcttgctgcgttcacactctctgcactacctcttcatgggttcctcagagcaggaccttggtctttccctgtttgaagctttgggctatgtggacgaccagctgttcgtgttctatgatcatgagagtcgccgtgtggagccccgaactccatgggtttccggtagaacgtcaagccagatgtggctgcagctgagtcagagtctgaaagggtgggatcacatgttcactgttgacttctggactattatggaaaatcacaaccacagcaaggagtcccacaccctgcaggtcatcctgggctgcaaaatgcaagaggacaacagtaccgagggcttctggaagtacgggtacgatgggcaggaccaccttgaattctgccctgacacactggattggagagcagcagaacccagggcctggcccaccaagctggagtgggaaaggcacaaaattcgggccaggcagaacagggcctacctcgagagggactgccctgtgcagctgcagcagttgctggagctggggagaggtgttttcgaccggccagtgaccactctacggtgtcgggccctgaactactacccccagaacatcaccatgaagtggctgaaggataggcagtcaatggatgccaaggaggtcgaacctaaagacgtattgcccaatggggatgggacctaccagggctggataaccttgactgtacccccaggggaagaacagagatatacttgccaggtggagcacccaggcctggatcagcccctccttgctttctgggagccctcaccatctagaactctagtcattggagtcatcagtggaattgctgtttttgtcatcatcttgttcattggaattttgttcataatattaaggaagaggcagacttcaagaggagtcatggggcactacgtcttagctgaacgtgagtgacacg 16 Mouse HFE -ctgagaggtctggaacctcagcaatggctacagggtgacttcttggatcctccacgt NM_010424.4ttccagatcctagtgaagaccggtggacccagctgaggacatgagcctatcagctgggctccctgtgcggccgctgctgctgctgctgctactgctgtggtccgtggccccgcaggcactgccaccgcgttcacattctctaagatacctcttcatgggtgcctcagagccagacctcgggctgcctttgtttgaggctaggggctatgtggatgaccagctctttgtgtcctacaatcatgagagtcgccgtgctgagcccagggccccgtggatcttggagcaaacctcaagccagctgtggctgcatctgagtcagagcctgaaagggtgggactacatgttcatagtagacttctggaccatcatgggcaactataaccacagtaaggtcacgaagttgggagtggtgtccgagtcccacatcctgcaggtggtcctaggctgtgaggtgcatgaagacaacagtaccagcggcttctggagatatggttatgacgggcaagatcacctggaattctgccccaagacactaaactggagcgcagccgagccaggggcctgggccaccaaggtggaatgggacgagcacaagatccgtgccaaacagaacagggactacctggagaaggactgccccgagcagctgaaacggctcctggagctggggagaggcgttctgggacagcaagtgcctactttggtgaaagtgactcgccactgggcctctacggggacctctctaaggtgtcaggctctggacttcttcccccagaacatcactatgaggtggttgaaggacaaccaaccactggatgccaaagatgtcaaccccgagaaggtgctacctaacggggatgagacctatcaaggctggctgacattggccgtggcccctggggacgagacaaggttcacctgtcaagtggagcacccaggcctggaccagcctctcactgcctcttgggagcccttgcaatctcaggccatgattatcggaatcatcagtggagtcaccgtctgtgccatcttcttggttggaattctgttcctaatcttaaggaaaaggaaggcttcaggaggaaccatgggtggctatgtcttaacagactgtgagtgatctgcagcctgctgaaccacggaagagagaaaactcagccaaagacttggagggggcacacttgctccactgtaggacacagttggacctaacacacagaaactgcctgagaactgtgctcttagctttctctgttcactttcttaaggtgttttctccagttaagttcagttcctgaatagtagtgattgcaccagttgcaacctctccctccagaactggtctcatgattcttaggctgcttcttggaagcatcctatgtttccttcatgcacctagactccatatgtctacgtaaagagcccctctaagtttagtggatacatgattcgtttccacatctgaagaagttgtgaaccttcatccggggatgctcacacatacttgagccagaatttttcacctatatcctagaatccaggacccactcaactatcctccatctgttatagagtgactcctctgtcaccatgccctgacttctctgccattggagtgttatatatatggatcatcaataaagccatgaaggc tacacaactgtg17 Rat HFE - tcagcaatggctacagggtgacttcttggatcctccacgtttccaggtcctagtgaaNM_001173435.1 aaccggtggacccagctggaggcatggaccgatcagctgggctccctgtgcggctgctattgctgctgctgttgttgctgctgtggtccgtggccccgcaggcgctgcggcccgtgcctactttggtgaaagtgactcgccactgggcctctacagggacctctctaaggtgtcaggctctgaatttcttcccccagaacatcactatgaggtggttgaaggacagccagcccctagatgccaaggatgtcaaccctgagaacgtgctgccaaatggggatgggacctatcagggctggctgaccttggctgtggcccctggagaagagacaaggttcagctgtcaagtggagcacccaggcctggatcagcctctcactgccacttgggagccctcacggtctcaggacatgattattggaatcataagtgggatcaccatttgtgccatcttctttgttggaattctgatcctagtcttaaggaaaaggaaggtttcaggaggaaccatgggtgactatgtcttaacagagtgtgagtgacctgcagcatgcagaagcacagaagagagaagactcagccaaagacttggaggggacacacttgctccattctagaacacagctggacctaacacacagaaactgcctgaggactctgcccttagctttcctgtttgctttcttaaggtgttttctccagttaagttcagttcctgaataatagtgactgccccagctgcaacctctcccttcagaaccagtctcatgatctttaagctgctacttgcaggcatccttcgttttctgcatccacctagacttcgtatgtctacttaaaaagccccactaaatttgggggacacatgattcatttccacatctgaagaagttatgaaccttcatcctgggatgcacacattcttgtgccagaatttttcatacatatcctaggacccattcaattgtcatttgagcctctctatctgttagtgactactctgacttctctgccattggagtgttatggcaataaagctatgaacgtta 18 Mouse BMPR1a -ccgcgcgagacgacgactgtacggccgcgcgaggggcgaccgggcccgggccgctgc NM_009758.4acgccgagggcggaggccgagccgggccccgccgccccgcggctgtccgtgcccgcccgcgccgagcgccggaggatgagtttctcgggatcccgatttatgaaaatatgcatcgctttgatactgtcttggaattcatgagatggaagcataggtcaaagctgttcggagaaattggaactacagttttatctagccacatctctgagaattctgaagaaagcagcaggtgaaagtcattgccaagtgattttgttctgtaaggaagcctccctcattcacttacaccagtgagacagcaggaccagtcattcaaagggccgtgtacaggacgcgtgcgaatcagacaatgactcagctatacacttacatcagattactgggagcctgtctgttcatcatttctcatgttcaagggcagaatctagatagtatgctccatggcactggtatgaaatcagacttggaccagaagaagccagaaaatggagtgactttagcaccagaggataccttgcctttcttaaagtgctattgctcaggacactgcccagatgatgctattaataacacatgcataactaatggccattgctttgccattatagaagaagatgatcagggagaaaccacattaacttctgggtgtatgaagtatgaaggctctgattttcaatgcaaggattcaccgaaagcccagctacgcaggacaatagaatgttgtcggaccaatttgtgcaaccagtatttgcagcctacactgccccctgttgttataggtccgttctttgatggcagcatccgatggctggttgtgctcatttccatggctgtctgtatagttgctatgatcatcttctccagctgcttttgctataagcattattgtaagagtatctcaagcaggggtcgttacaaccgtgatttggaacaggatgaagcatttattccagtaggagaatcattgaaagacctgattgaccagtcccaaagctctgggagtggatctggattgcctttattggttcagcgaactattgccaaacagattcagatggttcggcaggttggtaaaggccgctatggagaagtatggatgggtaaatggcgtggtgaaaaagtggctgtcaaagtgttttttaccactgaagaagctagctggtttagagaaacagaaatctaccagacggtgttaatgcgtcatgaaaatatacttggttttatagctgcagacattaaaggcactggttcctggactcagctgtatttgattactgattaccatgaaaatggatctctctatgacttcctgaaatgtgccacactagacaccagagccctactcaagttagcttattctgctgcttgtggtctgtgccacctccacacagaaatttatggtacccaagggaagcctgcaattgctcatcgagacctgaagagcaaaaacatccttattaagaaaaatggaagttgctgtattgctgacctgggcctagctgttaaattcaacagtgatacaaatgaagttgacatacccttgaataccagggtgggcaccaagcggtacatggctccagaagtgctggatgaaagcctgaataaaaaccatttccagccctacatcatggctgacatctatagctttggtttgatcatttgggaaatggctcgtcgttgtattacaggaggaatcgtggaggaatatcaattaccatattacaacatggtgcccagtgacccatcctatgaggacatgcgtgaggttgtgtgtgtgaaacgcttgcggccaatcgtgtctaaccgctggaacagcgatgaatgtcttcgagcagttttgaagctaatgtcagaatgttgggcccataatccagcctccagactcacagctttgagaatcaagaagacacttgcaaaaatggttgaatcccaggatgtaaagatttgacaattaaacaattttgagggagaatttagactgcaagaacttcttcacccaaggaatgggtgggattagcatggaataggatgttgacttggtttccagactccttcctctacatcttcacaggctgctaacagtaaaccttaccgtactctacagaatacaagattggaacttggaacttcaaacatgtcattctttatatatggacagctttgttttaaatgtggggtttttttgttttgctttttttgttttgttttggttttgatgcttttttggtttttatgaactgcatcaagactccaatcctgataagaagtctctggtcaacctctgggtactcactatcctgtccataaagtggtgctttctgtgaaagccttaagaaaattaatgagctcagcagagatggaaaaaggcatatttgccttctaccagagaaaacatctgtctgtgttctgtctttgtaaacagcctatagattatgatctctttgggatactgcctggtttatgatggtgcaccatacctttgatatgcataccagaattctctgctgccctagggcttagaagacaagaatgtaaaggttgcacaggaaggtatttgtggccagtggtttaaatatgcaatatctagttgacaatcgccaatttcataaaagccatccaccttgtaactgtagtaacttctccactgactttatttttagcataatagttgtgaaggccaaactccatgtaaagtgtccatagacttggactgttttcccccagtcaccattttgttctccttttggtaattatttttgttataaaaagccacctatccagaattggagctctctgtcttgaaccatactttgaaagaaacgcctcttccgtactgcatctgatcacaatgtgcatacctatgatcaaattctggagtctttgttctcggtacctcctaaaaaggaaagttgattcttgtgtaacatgcttttattttcagaacctgcacagctgtcattctagccatgttttacctacacactcagttctatacaagacagcccatacactctgtctcacatctgatccttggtgggaagtgttttaaagtagaactatgtatgaatttcagaattcatgcattttaaaacttcactaagatattgtctcatatctttatgagaatgtcagctgacttttcaactaacagtaaatgtattttagatatctaaatcttttgaaatttggttttacaatttctggtccctaattgtgaagacaagaggcagaagtacccagtcactacccatatttacactgaacgttattaaataaaatgatgtgtattttattataaaataaatataggccttgttatctcaaaaaacagatctggttcaaacttattataccaatatcatactatttaaatgttctaagtaaacaagccatgtgagcatcaagtggcattggctctttggatgaaacataaacttaaggtgattgtatcaacacatagagtgactgaaattaaatgggaggcaggtagagcatatgtccatctgtccacctacaggcatgactaaactacagctcatattccacaaatttgagatttgtcttgcctggtttgtttagtgagtctcatctgatgtacctaaagcctgagagtactgaggtctgattttatatctttcctgaataaactaaatcttttttgtcacttatcatcttaatgatatacctaaggaataattctttggcatgtttcagttgtgtgtggcagccactgtaatgactcttctctaagaaaggctgtcaggagttaattataaggcaggcagtgagcgctctagtcactgccttcccacgctgccatcactgcattcatgggaatcagtgacgttctcgaaatggcaaacgctgctgcttttccttatttggaatcctaaaatcaaaagttgcattaaacttactgtgttctcttctccctctcagccataaatgtaaaattcagtaagtaaaaatatttaaagagtgtatcagccctttggccagtgagatagctcagtagataaaggcatttgctgccaagtcctcaacctcaattcagactctgggggacacatggcgaagggaggagccaactaccccatcattgtcctctgacttccacacactccatggcttgcgcccctccccacagacacacaccatgtactccacaaaagtagtttaaaggaaaaaaagaatagaacccactgtgtaatggaataagtattatgtagttacttaacaacttgtaaaaatctggaaactatgatttggttcccctttgaatctagagtttaaaaaacagatggctaaaatcagccatcatttaaataattaaaaataaaagccccaaaccccaaactgcctaaataaataccaagtaatccaggaagccgtcatgtgtggtttgtatgaccagtagttctctggtacagagcatgttaagatttgccccagcctgattctctgaggtctctgcattactgagtactgttctgagtataaaatctgaactgatttctctagaaatactgtaacaaaaaggtattttagtcagcatgttatgttaacccttccactgtctagaaacttgaataagcacataaagacaccttttgctgtcatcatctgttgtcctggaatgtgccagttttaatttattcattctaatgatattcaatttgcttttcttttttagatgtttttcttgtttagagtaaaaggacgaatttttcaagaaccttgcatctctgatttggcctaaggtcaaattggatattgagtagtctattccagggcagatttcctaagcaatacttgtcttttcagctatgtattgttttgaaatgtttccatttcaacagaggtgtaagtcatgtgaaaagaaaggtggtgtagcccttgtggtaatgacacaagttgacttgcgtcagatgttaagcagggacagttctcccacctcctggctgtaaggagtggaaactaggcaagcagtgtatcagtccacagaggacaggaagggtcatcccataaagaaagcctgtgagtatggctttggcaaaaaattagacataatactgtccttttaggttgtgctctgttctttcctttcagtggaattatttaagctctttagtggcctttgtttttcccacttaaaaactaaaatgtagcatatattgtataaaatggaaatattaatagcttagggaaactgtacataaggcattgacaggtttaaaaaaagcatttttattatgcagttgtaaaacaccaaaaatatagattcatcttgatatgtaacactaagtgtattttgtacagcatctgatttgaaaggtgccttatgaagtttaccattaattgctttgttctatatacagattatgtccaatgtatcatttttcagtaaataacctt attttagta 19Rat BMPR1a - gaattcatgagatggaaacataggtcaaagctgtttggagaaattggaactacagttNM_030849.1 ttatctagccacatctctgagaagtctgaagaaagcagcaggtgaaagtcattgtcaagtgattttgttcttctgtaaggaaacctcgttcagtaaggccgtttacttcagtgaaacagcaggaccagtaatcaaggtggcccggacaggacacgtgcgaattggacaatgactcagctatacacttacatcagattactgggagcctgtctgttcatcatttctcatgttcaagggcagaatctagatagtatgctccatggtactggtatgaaatcagacgtggaccagaagaagccggaaaatggagtgacgttagcaccagaggacaccttacctttcttaaaatgctattgctcaggacactgcccagatgacgctattaataacacatgcataactaatggccattgctttgccattatagaagaagatgatcagggagaaaccacgttaacttctgggtgtatgaagtatgaaggctctgattttcaatgcaaggattcaccaaaagcccagctacgcaggacaatagaatgttgtcggaccaatttgtgcaaccaatatttgcagcctacactgccccctgtcgttataggcccattctttgatggcagcgtccgatggctggctgtgctcatctctatggctgtctgtattgtcgccatgatcgtcttctccagctgcttctgttacaaacattactgtaagagtatctcaagcagaggtcgttacaaccgtgacttggaacaggatgaagcatttattccagtaggagaatcactgaaagacctgattgaccagtcacaaagctctggtagtggatctggattacctttattggttcagcgaactattgccaaacagattcagatggttcggcaggttggtaaaggccggtatggagaagtatggatgggtaaatggcgtggtgaaaaagtggctgtcaaagtattttttaccactgaagaagctagctggtttagagaaacagaaatctaccagacggtgttaatgcgtcatgaaaatatacttggttttatagctgcagacattaaaggcaccggttcctggactcagctgtatttgattactgattaccatgagaatgggtctctctatgacttcctgaaatgtgccaccctggacaccagagccctactcaagttagcttattctgctgcctgtggtctgtgccacctccacacagaaatttatggcacgcaaggcaagcctgcaattgctcatcgagacctgaagagcaaaaacatccttattaagaaaaatggtagttgctgtattgctgacctgggcctagctgttaaattcaacagtgacacaaatgaagttgacatacccttgaacaccagggtgggcaccaggcggtacatggctccagaagtgctggacgagagcctgagtaaaaaccatttccagccctacatcatggctgacatctacagctttggtttgatcatttgggagatggcccgtcgctgtattacaggaggaatcgtggaggaatatcaattaccatattacaacatggtgcctagtgacccatcttatgaagacatgcgtgaggtcgtgtgtgtgaaacgcttgcggccaatcgtctctaaccgctggaacagtgatgaatgtcttcgagccgttttgaagctgatgtcagaatgctgggcccataatccagcatccagactcacagctttgagaatcaagaagacgctcgcaaagatggttgaatcccaggatgtaaagatttgacaaacagttttgagaaagaatttagactgcaagaaattcacccgaggaagggtggagttagcatggactaggatgtcggcttggtttccagactctctcctctacatcttcacaggctgctaacagtaaactttcaggactctgcagaatgcagggttggagcttcagacataggacttcagacatgctgttctttgcgtatggacagctttgttttaaatgtgggcttttgatgcctttttggtttttatgaattgcatcaagactccaatcctgataagaagtctctggtcaaactctggttactcactatcctgtccataaagtggtgctttctgtgaaagccttaaggaaattagtgagctcagcagagatggagaaaggcatatttgccctctacagagaaaatatctgtctgtgttctgtctctgtaaacagcctggactatgatctctttgggatgctgcctggttgatgatggtgcatcatgcctctgatatgcataccagacttcctctgctgccatgggcttacaagacaagaatgtgaaggttgcacaggacggtatttgtggccagtggtttaaatatgcaatatctaatcgacattcgccaatctcataaaagccatctaccttgtaactgaagtaacttctctaccaactttatttttagcataatagttgtaaaggccaaactatgtataaagtgtccatagactcgaactgttttcctccagtcaccattttgttttccttttggtaattatttttgttatataattcctcctatccagaattggcgctcactgtcttgaaccatactttgaaagaaatgcctcttcctggagtctgccttactgcatctgatcaccatgtgcatacctctgatcaaattctggagtctttgttctcggtacctcttaaaaagggaaattgtgtatcatgtgtagtgtgcttttattttcaaaatcttcatagcctttattctagccatttttacctacatactcattctgtacaaaacagctcactcggtctcacggctgatcctcagtggaaatgatttaaagtagagctgtgtacgaatttcagaattcatgtatttaaaaacttcacactaacactttactaagatattgtctcatatcttttatgaggatgtcagctgattttcaatgactataaatgtatcttagctatctaaatcttttgaaatttggttttataatttctggtccctaacttgtgaagacaaagaggcagaagtacccagtctaccacatttacactgtacattattaaataaaaaaatgtatattttaaaaaaaaaaaaaaaaaaaaa 20 Human SMAD4 -atgctcagtggcttctcgacaagttggcagcaacaacacggccctggtcgtcgtcgc NM_005359.5cgctgcggtaacggagcggtttgggtggcggagcctgcgttcgcgccttcccgctctcctcgggaggcccttcctgctctcccctaggctccgcggccgcccagggggtgggagcgggtgaggggagccaggcgcccagcgagagaggccccccgccgcagggcggcccgggagctcgaggcggtccggcccgcgcgggcagcggcgcggcgctgaggaggggcggcctggccgggacgcctcggggcgggggccgaggagctctccgggccgccggggaaagctacgggcccggtgcgtccgcggaccagcagcgcgggagagcggactcccctcgccaccgcccgagcccaggttatcctgaatacatgtctaacaattttccttgcaacgttagctgttgtttttcactgtttccaaaggatcaaaattgcttcagaaattggagacatatttgatttaaaaggaaaaacttgaacaaatggacaatatgtctattacgaatacaccaacaagtaatgatgcctgtctgagcattgtgcatagtttgatgtgccatagacaaggtggagagagtgaaacatttgcaaaaagagcaattgaaagtttggtaaagaagctgaaggagaaaaaagatgaattggattctttaataacagctataactacaaatggagctcatcctagtaaatgtgttaccatacagagaacattggatgggaggcttcaggtggctggtcggaaaggatttcctcatgtgatctatgcccgtctctggaggtggcctgatcttcacaaaaatgaactaaaacatgttaaatattgtcagtatgcgtttgacttaaaatgtgatagtgtctgtgtgaatccatatcactacgaacgagttgtatcacctggaattgatctctcaggattaacactgcagagtaatgctccatcaagtatgatggtgaaggatgaatatgtgcatgactttgagggacagccatcgttgtccactgaaggacattcaattcaaaccatccagcatccaccaagtaatcgtgcatcgacagagacatacagcaccccagctctgttagccccatctgagtctaatgctaccagcactgccaactttcccaacattcctgtggcttccacaagtcagcctgccagtatactggggggcagccatagtgaaggactgttgcagatagcatcagggcctcagccaggacagcagcagaatggatttactggtcagccagctacttaccatcataacagcactaccacctggactggaagtaggactgcaccatacacacctaatttgcctcaccaccaaaacggccatcttcagcaccacccgcctatgccgccccatcccggacattactggcctgttcacaatgagcttgcattccagcctcccatttccaatcatcctgctcctgagtattggtgttccattgcttactttgaaatggatgttcaggtaggagagacatttaaggttccttcaagctgccctattgttactgttgatggatacgtggacccttctggaggagatcgcttttgtttgggtcaactctccaatgtccacaggacagaagccattgagagagcaaggttgcacataggcaaaggtgtgcagttggaatgtaaaggtgaaggtgatgtttgggtcaggtgccttagtgaccacgcggtctttgtacagagttactacttagacagagaagctgggcgtgcacctggagatgctgttcataagatctacccaagtgcatatataaaggtctttgatttgcgtcagtgtcatcgacagatgcagcagcaggcggctactgcacaagctgcagcagctgcccaggcagcagccgtggcaggaaacatccctggcccaggatcagtaggtggaatagctccagctatcagtctgtcagctgctgctggaattggtgttgatgaccttcgtcgcttatgcatactcaggatgagttttgtgaaaggctggggaccggattacccaagacagagcatcaaagaaacaccttgctggattgaaattcacttacaccgggccctccagctcctagacgaagtacttcataccatgccgattgcagacccacaacctttagactgaggtcttttaccgttggggcccttaaccttatcaggatggtggactacaaaatacaatcctgtttataatctgaagatatatttcacttttgttctgctttatcttttcataaagggttgaaaatgtgtttgctgccttgctcctagcagacagaaactggattaaaacaattttttttttcctcttcagaacttgtcaggcatggctcagagcttgaagattaggagaaacacattcttattaattcttcacctgttatgtatgaaggaatcattccagtgctagaaaatttagccctttaaaacgtcttagagccttttatctgcagaacatcgatatgtatatcattctacagaataatccagtattgctgattttaaaggcagagaagttctcaaagttaattcacctatgttattttgtgtacaagttgttattgttgaacatacttcaaaaataatgtgccatgtgggtgagttaattttaccaagagtaactttactctgtgtttaaaaagtaagttaataatgtattgtaatctttcatccaaaatattttttgcaagttatattagtgaagatggtttcaattcagattgtcttgcaacttcagttttatttttgccaaggcaaaaaactcttaatctgtgtgtatattgagaatcccttaaaattaccagacaaaaaaatttaaaattacgtttgttattcctagtggatgactgttgatgaagtatacttttcccctgttaaacagtagttgtattcttctgtatttctaggcacaaggttggttgctaagaagcctataagaggaatttcttttccttcattcatagggaaaggttttgtattttttaaaacactaaaagcagcgtcactctacctaatgtctcactgttctgcaaaggtggcaatgcttaaactaaataatgaataaactgaatattttggaaactgctaaattctatgttaaatactgtgcagaataatggaaacattacagttcataataggtagtttggatatttttgtacttgatttgatgtgactttttttggtataatgtttaaatcatgtatgttatgatattgtttaaaattcagtttttgtatcttggggcaagactgcaaacttttttatatcttttggttattctaagccctttgccatcaatgatcatatcaattggcagtgactttgtatagagaatttaagtagaaaagttgcagatgtattgactgtaccacagacacaatatgtatgctttttacctagctggtagcataaataaaactgaatctcaacatacaaagttgaattctaggtttgatttttaagattttttttttcttttgcacttttgagtccaatctcagtgatgaggtaccttctactaaatgacaggcaacagccagttctattgggcagctttgtttttttccctcacactctaccgggacttccccatggacattgtgtatcatgtgtagagttggttttttttttttttaatttttattttactatagcagaaatagacctgattatctacaagatgataaatagattgtctacaggataaatagtatgaaataaaatcaaggattatctttcagatgtgtttacttttgcctggagaacttttagctatagaaacacttgtgtgatgatagtcctccttatatcacctggaatgaacacagcttctactgccttgctcagaaggtcttttaaatagaccatcctagaaaccactgagtttgcttatttctgtgatttaaacatagatcttgatccaagctacatgacttttgtctttaaataacttatctaccacctcatttgtactcttgattacttacaaattctttcagtaaacacctaattttcttctgtaaaagtttggtgatttaagttttattggcagttttataaaaagacatcttctctagaaattgctaactttaggtccattttactgtgaatgaggaataggagtgagttttagaataacagatttttaaaaatccagatgatttgattaaaaccttaatcatacattgacataattcattgcttcttttttttgagatatggagtcttgctgtgttgcccaggcaggagtgcagtggtatgatctcagctcactgcaacctctgcctcccgggttcaactgattctcctgcctcagcctccctggtagctaggattacaggtgcccgccaccatgcctggctaacttttgtagttttagtagagacggggttttgcctgttggccaggctggtcttgaactcctgacctcaagtgatccatccaccttggcctcccaaagtgctgggattacgggcgtgagccactgtccctggcctcattgttcccttttctactttaaggaaagttttcatgtttaatcatctggggaaagtatgtgaaaaatatttgttaagaagtatctctttggagccaagccacctgtcttggtttctttctactaagagccataaagtatagaaatacttctagttgttaagtgcttatatttgtacctagatttagtcacacgcttttgagaaaacatctagtatgttatgatcagctattcctgagagcttggttgttaatctatatttctatttcttagtggtagtcatctttgatgaataagactaaagattctcacaggtttaaaattttatgtctactttaagggtaaaattatgaggttatggttctgggtgggttttctctagctaattcatatctcaaagagtctcaaaatgttgaatttcagtgcaagctgaatgagagatgagccatgtacacccaccgtaagacctcattccatgtttgtccagtgcctttcagtgcattatcaaagggaatccttcatggtgttgcctttattttccggggagtagatcgtgggatatagtctatctcatttttaatagtttaccgcccctggtatacaaagataatgacaataaatcactgccatataaccttgctttttccagaaacatggctgttttgtattgctgtaaccactaaataggttgcctataccattcctcctgtgaacagtgcagatttacaggttgcatggtctggcttaaggagagccatacttgagacatgtgagtaaactgaactcatattagctgtgctgcatttcagacttaaaatccatttttgtggggcagggtgtggtgtgtaaaggggggtgtttgtaatacaagttgaaggcaaaataaaatgtcctgtctcccagatgatatacatcttattatttttaaagtttattgctaattgtaggaaggtgagttgcaggtatctttgactatggtcatctggggaaggaaaattttacattttactattaatgctccttaagtgtctatggaggttaaagaataaaatggtaaatgtttctgtgcctggtttgatggtaactggttaatagttactcaccattttatgcagagtcacattagttcacaccctttctgagagccttttgggagaagcagttttattctctgagtggaacagagttctttttgttgataatttctagtttgctcccttcgttattgccaactttactggcattttatttaatgatagcagattgggaaaatggcaaatttaggttacggaggtaaatgagtatatgaaagcaattacctctaaagccagttaacaattattttgtaggtggggtacactcagcttaaagtaatgcatttttttttcccgtaaaggcagaatccatcttgttgcagatagctatctaaataatctcatatcctcttttgcaaagactacagagaataggctatgacaatcttgttcaagcctttccatttttttccctgataactaagtaatttctttgaacataccaagaagtatgtaaaaagtccatggccttattcatccacaaagtggcatcctaggcccagccttatccctagcagttgtcccagtgctgctaggttgcttatcttgtttatctggaatcactgtggagtgaaattttccacatcatccagaattgccttatttaagaagtaaaacgttttaatttttagcctttttttggtggagttatttaatatgtatatcagaggatatactagatggtaacatttctttctgtgcttggctatctttgtggacttcaggggcttctaaaacagacaggactgtgttgcctttactaaatggtctgagacagctatggttttgaatttttagttttttttttttaacccacttcccctcctggtctcttccctctctgataattaccattcatatgtgagtgttagtgtgcctccttttagcattttcttcttctctttctgattcttcatttctgactgcctaggcaaggaaaccagataaccaaacttactagaacgttctttaaaacacaagtacaaactctgggacaggacccaagacactttcctgtgaagtgctgaaaaagacctcattgtattggcatttgatatcagtttgatgtagcttagagtgcttcctgattcttgctgagtttcaggtagttgagatagagagaagtgagtcatattcatattttcccccttagaataatattttgaaaggtttcattgcttccacttgaatgctgctcttacaaaaactggggttacaagggttactaaattagcatcagtagccagaggcaataccgttgtctggaggacaccagcaaacaacacacaacaaagcaaaacaaaccttgggaaactaaggccatttgttttgttttggtgtcccctttgaagccctgccttctggccttactcctgtacagatatttttgacctataggtgcctttatgagaattgagggtctgacatcctgccccaaggagtagctaaagtaattgctagtgttttcagggattttaacatcagactggaatgaatgaatgaaactttttgtcctttttttttctgtttttttttttctaatgtagtaaggactaaggaaaacctttggtgaagacaatcatttctctctgttgatgtggatacttttcacaccgtttatttaaatgctttctcaataggtccagagccagtgttcttgttcaacctgaaagtaatggctctgggttgggccagacagttgcactctctagtttgccctctgccacaaatttgatgtgtgacctttgggcaagtcatttatcttctctgggccttagttgcctcatctgtaaaatgagggagttggagtagattaattattccagctctgaaattctaagtgaccttggctaccttgcagcagttttggatttcttccttatctttgttctgctgtttgagggggctttttacttatttccatgttattcaaaggagactaggcttgatattttattactgttcttttatggacaaaaggttacatagtatgcccttaagacttaattttaaccaaaggcctagcaccaccttaggggctgcaataaacacttaacgcgcgtgcgcacgcgcgcgcgcacacacacacacacacacacacacacacacaggtcagagtttaaggctttcgagtcatgacattctagcttttgaattgcgtgcacacacacacgcacgcacacactctggtcagagtttattaaggctttcgagtcatgacattatagcttttgagttggtgtgtgtgacaccaccctcctaagtggtgtgtgcttgtaattttttttttcagtgaaaatggattgaaaacctgttgttaatgcttagtgatattatgctcaaaacaaggaaattcccttgaaccgtgtcaattaaactggtttatatgactcaagaaaacaataccagtagatgattattaactttattcttggctctttttaggtccattttgattaagtgacttttggctggatcattcagagctctcttctagcctacccttggatgagtacaattaatgaaattcatattttcaaggacctgggagccttccttggggctgggttgagggtggggggttggggagtcctggtagaggccagctttgtggtagctggagaggaagggatgaaaccagctgctgttgcaaaggctgcttgtcattgatagaaggactcacgggcttggattgattaagactaaacatggagttggcaaactttcttcaagtattgagttctgttcaatgcattggacatgtgatttaagggaaaagtgtgaatgcttatagatgatgaaaacctggtgggctgcagagcccagtttagaagaagtgagttgggggttggggacagatttggtggtggtatttcccaactgtttcctcccctaaattcagaggaatgcagctatgccagaagccagagaagagccactcgtagcttctgctttggggacaactggtcagttgaaagtcccaggagttcctttgtggctttctgtatacttttgcctggttaaagtctgtggctaaaaaatagtcgaacctttcttgagaactctgtaacaaagtatgtttttgattaaaagagaaagccaactaaaaaaaaa aaaaaaaaaaa 21Mouse SMAD4 - ccgctgcggtaacggagcggctcgggtggcggagcccgtgttcgcgtccgtccgcccNM_008540.2 gcccgcccgccgtcctccggaggcccttcccgcgccgcgctccgctccgcggccgtccccggggcgggagcgcgtgaccggagccggcgcccgcgagcgaggccccccgcagcggggcggctccggagctccagcggcccggccggccggcgcggtccgcggcgcggcggggagagggggccgcctgggccggacgccgcgggcggggcccgggaagcgacagcgaggcgaggcgcggtgcggcgcggagcccaggtcatcctgctcaccagatgtcttgacagtttttcttgcaacattggccattggttttcactgccttcaaaagatcaaaattactccagaaattggagagttggatttaaaagaaaaaacttgaacaaatggacaatatgtctataacaaatacaccaacaagtaacgatgcctgtctgagcattgtacatagtttgatgtgtcatagacaaggtggggaaagtgaaacctttgcaaaaagagcaattgagagtttggtaaagaagctgaaagagaaaaaagatgaattggattctttaataacagctataactacaaatggagctcatcctagcaagtgtgtcaccatacagagaacattggatggacgacttcaggtggctggtcggaaaggatttcctcatgtgatctatgcccgtctgtggaggtggcctgatctacacaagaatgaactaaagcatgttaaatattgtcagtatgcgtttgacttaaaatgtgacagtgtctgtgtgaatccatatcactatgagcgggttgtctcacctggaattgatctctcaggattaacactgcagagtaatgctccaagtatgttagtgaaggatgagtacgttcacgactttgaaggacagccgtccttacccactgaaggacattcgattcaaaccatccaacacccgccaagtaatcgcgcatcaacggagacgtacagcgccccggctctgttagccccggcagagtctaacgccaccagcaccaccaacttccccaacattcctgtggcttccacaagtcagccggccagtattctggcgggcagccatagtgaaggactgttgcagatagcttcagggcctcagccaggacagcagcagaatggatttactgctcagccagctacttaccatcataacagcactaccacctggactggaagtaggactgcaccatacacacctaatttgcctcaccaccaaaacggccatcttcagcaccacccgcctatgccgccccatcctggacattactggccagttcacaatgagcttgcattccagcctcccatttccaatcatcctgctcctgagtactggtgctccattgcttactttgaaatggacgttcaggtaggagagacgtttaaggtcccttcaagctgccctgttgtgactgtggatggctatgtggatccttcgggaggagatcgcttttgcttgggtcaactctccaatgtccacaggacagaagcgattgagagagcgaggttgcacataggcaaaggagtgcagttggaatgtaaaggtgaaggtgacgtttgggtcaggtgccttagtgaccacgcggtctttgtacagagttactacctggacagagaagctggccgagcacctggcgacgctgttcataagatctacccaagcgcgtatataaaggtctttgatctgcggcagtgtcaccggcagatgcagcaacaggcggccactgcgcaagctgcagctgctgctcaggcggcggccgtggcagggaacatccctggccctgggtccgtgggtggaatagctccagccatcagtctgtctgctgctgctggcatcggtgtggatgacctccggcgattgtgcattctcaggatgagctttgtgaagggctggggcccagactaccccaggcagagcatcaaggaaaccccgtgctggattgagattcaccttcaccgagctctgcagctcttggatgaagtcctgcacaccatgcccattgcggacccacagcctttagactgagatctcacaccacggacgccctaaccatttccaggatggtggactatgaaatatactcgtgtttataatctgaagatctattgcattttgttctgctctgtcttttcctaaagggttgagagatgtgtttgctgccttgctcttagcagacagaaactgaattaaaacttcttttctattttagaactttcaggtgtggctcagtgcttgaagatcagaaagatgcagttcttgctgagtcttccctgctggttctgtatggaggagtcggccagtgctgggcgctcagccctttagtgtgtgcgagcgccttgcatgccgaggagagtcagagctgctgattgtaaggctgagaagttctcacagttaagccacctgccccttagtgggcgagttattaaacgcactgtgctcacgtggcgctgggccagccagctctaccaagagcaactttactctcctttaaaaaccttttagcaacctttgattcacaatggtttttgcaagttaaacagtgaaggtgaattaaattcatactgtcttgcagacttcagggtttcttccccaagacaaaacactaatctgtgtgcatattgacaattccttacaattatcagtcaaagaaatgccatttaaaattacaatttttttaatccctaatggatgaccactatcaagatgtatactttgccctgttaaacagtaaatgaattcttctatatttctaggcacaaggttagttatttaaaaaaaaaaaaaaaagcctaggggagggatttttcccttaattcctagggagaaggttttgtataaaacactaaaagcagtgtcactctgcctgctgcttcactgttctgcaaggtggcagtacttcaactgaaataatgaatattttggaaactgctaaattctatgttaaatactgtgcagaataatggaaacagtgcagttggtaacaggtggtttggatatttttgtacttgatttgatgtgtgacttcttttcatatactgttaaaatcatgtatgttttgacattgtttaaaattcagtttttgtatcttagggcaagactgcagacttttttataccttttggttataagccctgtgtttgccatccttgatcacttggcggtgactttgtagagattgaagtggaggagttaagacacattgactgtaccacagacacacatgtatactttctacctagttactagcgtaaataaaactgagtcactataaaaaaaaaaaaaaaaaaaaa 22 Mouse IL6R -gcagtgcgagctgagtgtggagcccgaggccgagggcgactgctctcgctgccccag NM_010559.2tctgccggccgcccggccccggctgcggagccgctctgccgcccgccgtcccgcgtagaaggaagcatgctgaccgtcggctgcacgctgttggtcgccctgctggccgcgcccgcggtcgcgctggtcctcgggagctgccgcgcgctggaggtggcaaatggcacagtgacaagcctgccaggggccaccgttaccctgatttgccccgggaaggaagcagcaggcaatgttaccattcactgggtgtactctggctcacaaaacagagaatggactaccacaggaaacacactggttctgagggacgtgcagctcagcgacactggggactatttatgctccctgaatgatcacctggtggggactgtgcccttgctggtggatgttcccccagaggagcccaagctctcctgcttccggaagaacccccttgtcaacgccatctgtgagtggcgtccgagcagcaccccctctccaaccacgaaggctgtgctgtttgcaaagaaaatcaacaccaccaacgggaagagtgacttccaggtgccctgtcagtattctcagcagctgaaaagcttctcctgccaggtggagatcctggagggtgacaaagtataccacatagtgtcactgtgcgttgcaaacagtgtgggaagcaagtccagccacaacgaagcgtttcacagcttaaaaatggtgcagccggatccacctgccaaccttgtggtatcagccatacctggaaggccgcgctggctcaaagtcagctggcagcaccctgagacctgggacccgagttactacttgctgcagttccagcttcgataccgacctgtatggtcaaaggagttcacggtgttgctgctcccggtggcccagtaccaatgcgtcatccatgatgccttgcgaggagtgaagcacgtggtccaggtccgtgggaaggaggagcttgaccttggccagtggagcgaatggtccccagaggtcacgggcactccttggatagcagagcccaggaccaccccggcaggaatcctctggaaccccacacaggtctctgttgaagactctgccaaccacgaggatcagtacgaaagttctacagaagcaacgagtgtcctcgccccagtgcaagaatcctcgtccatgtccctgcccacattcctggtagctggaggaagcttggcgtttgggttgcttctctgtgtcttcatcatcctgagactcaagcagaaatggaagtcagaggctgagaaggaaagcaagacgacctctcctccacccccaccgtattccttgggcccactgaagccgaccttccttctggttcctctcctcaccccacacagctctgggtctgacaataccgtaaaccacagctgcctgggtgtcagggacgcacagagcccttatgacaacagcaacagagactacttattccccagataatcatctggatggtacctggcagctggcagggcaccacgagatcagcacacaagtttctcatgcgggtcccatccacctggggtggggtggggcgggcggggctgcagcttcactaacccacaagagctctgcacaggttctgagtaggtgcagctggtgctgcataggctctgaaggaaggaaggggctgtgaggaacacaggccattgtgaagacagcttgtgatgactgaatagagatgcccgtcagctccacatctgatagtggctcacaagctgcaccctcaggaggcctcagaaaggggctccaaaggctgccccagctgcctcgctctgcctcactgccccaagccaccttttagctctcgaactcctaaagtccaagcactttgccattctctttccgaggccactgaggccgggtggaagcttggttccgatttccttctcaacatctggaaagcagctgggcccggtggtggtgactaatatctcagggcctgatggtttacgcgagtgacaatttctcacaagcagtttttaaatgtgaatgatgaccccaggcactgctggctgcggaggcttcattttcctcttcgatctcaggacttcaggcgaaaagcggagtggaagtagagagcggatgggtgtccaccgtcctcatggtacttgcgggaggtacagcctggaaaacacgtttcctgtccccctactctcccaggagagggatgatggtagggggtgcctcttccagggcggagagaactactttaccccagccttgcccattctgatttcaactggactggagctactaggaaagtcgacattcatgcaaaaagaaaaaacgttaactagcaagaatgcactttcattttggtttttagagaactgttgcctgtttctctcaagagtctggaagaggccgctcactgcacactactgtatgaaccctcactgcccaccctggaggaccaagtgcagtaacggtagcccaaacaccaagtcaagtgaaaatcgagggaaaaaaaaaacaaacaagcaacaaaaaaaaaaaaccaaaactaaactaaaaaacaaatcacccccccaaaaaaaaacaaaaccaaaaaccaaaaaaaacaaaaaaacaaaacaacaacaacaaaaaaaacccaaaccaacccgctgtttcctataacagaaaagcctttggtttcattttttattttgatttttttgtcttaaaaagtataaaaatagcctgtccatgctctgcttcagggaatgagcctgtgaacactcccaggcgcaggcaggaagggtgtctgcttcctgctacacctcactgccaccttggccttccttgctttacgtttgactgagtggcctcagatgctttcccctggggctttgaggaatccagtgatgttagtggtcaccgaggagaccacagagccacagtgtggtgcttagattaaagtgacttctgcaaccacagcaccccacacctgccgtcttactgaactatgccagtaacttgccttttctgccaccaccacgagacgagacgggcagagctcggaagctgtcaccccatgccctctgcttgtccgctctaggggccactgacctaagcattagttattttattttattttatttttttgtgggttttgtacattttaggtcctgttgctgtcttagaaaaggctctgtaggttgacagaaaatcaggccaagtattcatgttttgttttttttttttttccttctttcctcctttgctaagtttttgggactcaagggtagcaaaactgctgtgaaagggaaatttattaaaaatgttacagatcgtg 23 Rat IL6R -gccccacgtagaaggaaccatgctggccgtcggctgcaccctgctggtcgccctgct NM_017020.3ggccgcgcccgcagtcgcgctggtccttgggagctgccgcgcgctggaggtggcaaatggtacggtgacgagcctgccaggggccactgttaccctgatctgccctgggaaggaagcagcaggcaatgctaccattcactgggtgtactcaggctcacagagcagagaatggactaccacgggaaacacactggttctgagggccgtgcaggtcaacgacactgggcactatttgtgcttcctggatgatcatctggttgggactgtgcccttgctggtggatgttcccccagaggagcccaagctctcctgcttccggaagaacccccttgtaaatgccttttgtgagtggcatccaagcagcactccctctccaaccacgaaggctgtgatgtttgcaaagaaaatcaacaccaccaatgggaagagtgacttccaggtgccttgccagtattctcagcagctgaaaagcttctcctgcgaggtggagatcctggagggtgacaaagtgtaccacatagtgtcactgtgcgttgcaaacagtgtcggaagcaggtccagccacaatgtagtatttcagagtttaaaaatggtgcagccggatccacctgccaaccttgtggtatcagccatacctggaaggcctcgttggctcaaagtcagttggcaagaccctgagtcctgggacccaagttactacttgttgcaattcgagcttcgataccgacctgtatggtcaaagacgttcacggtgtggccgctccaggtggcccagcatcaatgtgtcatccatgatgccttgcgaggagtaaagcatgtggtgcaggtccgagggaaggaggagtttgacattggccagtggagcaaatggtccccggaggtcacaggcactccttggctagcagagcccaggaccactccggcagggatcccggggaaccccacacaggtctctgttgaagactatgacaaccacgaggatcagtacggaagttctacagaagcaacgagtgtcctcgccccagtgcaaggatcctcgcctatacccctgcccacattcctggtagctggaggaagcctggcgtttggattgcttctctgtgtcttcatcatcttgagactcaagaagaaatggaagtcacaggctgagaaggaaagcaagacgacttctcccccaccgtatcccttgggaccgctgaagccgaccttcctcctggttcctctcctcaccccatcagggtcccataacagctctgggactgacaacaccggaagccacagctgcctgggtgtcagggacccacagtgccctaatgacaacagcaacagagactacttattccccagataattgtctggagggtacctggcagctggcacgcaagtttctcactgccggccccgtccaccagggctgggggcggggtgggcggggctgcagcttcacgatcccacaggagccttgcaaaggttctgagtgggagaagactggtgtgctgcacgggcttcgaaagaaggggctgtgaggagcacgagccatcatgaagagagcctgtgatgactctgaatagagacgcccgcccatcagctacacacctgatggtggctctcaagctatcctctcaggaagcctctgggaggggcgacaaaggctgccccagttgcctagctctggctcactggcccaagctgccttttagcttgaactcctaaaatccaagcaccttggccattctcttcctaggccaccgaggccgcggggaagcttggttctactttccttctcaacacctggagaagcagctgcccggtggtggtgactaacgtatcagggcctgatggcttatgaggaatgacaattaattcctcataagcagtttttaaatgtgaatagtaatcctaggcactgctgacttgaggttttattttcttcaatctcaggacttcaggagagaagcagagcagaagtagagagaggatgggtgtccattgtccgtgtggtacttgagggggatacagcctggaaaacacgtttcctgtccccctactctcccagaagaggtagggggtggcgcctcttccagggcagagagtataactactttacctggccttgcccatactggtttcaactggacttgagctactaggaaaaatgacattcatgcaaaaagaaaactttaactagcaagaatgcacttccactttggtttctagaggactgttgctcctcttgagacgctggaagaggccgctcactgtaccctggtgtatgagccctcaccccccaccccagggtaagtgcagtaactttagtctaaacaccgagtcaggtaaaaatcgaggaaaaaacaaccctgtttcctgtaacagaaaagcctttggtttcgttttgtattttgattttttttttgtcttaaaaagtgtaaaaatagtctgtccatactctgcttcagggaatgacctgtgaatactccccaggcgtgggcaggaagggtgtctgcttcctgctacacctcactgccacctcggccttccttgctttacattcaactgagttgcctcagctgctttcccctggggcgctgaaaaagccagtgatgttggtggtcaccgagaagaccacagagccacagagtaatgctgtgattgaagcgagttacgcaaccacagcaccccacatttgctgtattatagaactatgctaggagcttgccttttcacaaaataccaccaccacgagacgtggcagagctcggaagctgtcaccttgtgccatctgcttgccagctccaaggggccactgacttaagcagttattttctttgtgggctttgttcatttcagggcctgttgctgtcttagaaaaagctctgtcggttgacaaaaacatcagacaggtagtcatgtttatttattttttttccttctttgctaagtctttgggactcaagggtagtaaaaaatgctgtgaaaagggaaacattagaaacagcgatcttcggggaataggtgactgtgcccacgcactgttcttcagtccctcacgtggctctgcccgagtgctgttccaagccaggcagagcaggctggcggaagattgaaatccagatagttcgttatctctgagagctaaatagctttgatctccaagctgttattgctttcactattgtaacaggatagcctcccccccccatgtcaaaaggatgcttttcccttttgactttttataagctaagtcagtgaagtctgtttcatctgagctccagcttcgttcagttcgcacaggtgtatgccctcagctgcttcgggcctcagatctgtgctagttgaatggttgtcccatccttgggtcatccttaccagagtttctgcagcccacaggtctgccttgtcaacagtaccacttaacaccagcattcagtgcccaggcagccagatgtggagggtttacccagagatgatttaaacatgaccttaaacgtgtatggtagaacgaggggaacccataccagctcaggttctaaagagatctttgattcttctggcattagtgaaatagctttaaactatttcaaggaagaagccttggccacacccacgacatttggtgacaatcctttctctccatgagccttgtctttacaccttctcacctggctgaaagctcacactgaatctttcctatgtccctggtgtcttgggagaaaggaaactggtatgggcttcactgctggaattggcttggagccagcgtgtggcgcagcgcctggcagggtgggccaggcttagttatggtgtgctggtttaaggaatgcctggcttgcctggttgcttgggttctgagctgcagagtttcctagcagttctttatggctgacctagttggggaagattcccacactcaactgcaggtggaggtggtgagaaagctgttttcatttggagaggcaggatcagcccaagaagctttcagtgggagagcctacagtgaggctgtacctcactgtgggaggaggcaggccagctggctcaggtcctgggactggcactggggagggtctgccaaaggtccctccagcctgtagtcctagcatagtcgggtgccagttccaggaagtttctatggcaaccttagtgctcattaaggaacattgtcagttttgtgaacatatgctcagatggagatcttgttttcagagaaaggactggtacagtgtgtaacaagctggagcagacagagagactttttggcaagagatcacatccgttaagcagaatacctcagtgctacatgtttttgtctttgagacaatgtttttaaggtttttatgctctgttacctgtaagctgatacctaaaactttctgcaaagtcagggtttttcaatgccttttttttttttttgccattgtttgctttaaagtgaagattgtaactgtttgaaataaataatttctaaaactgca 24 Human BMP6 -caactgggggcgccccggacgaccatgagagataaggactgagggccaggaagggga NM_001718.4agcgagcccgccgagaggtggcggggactgctcacgccaagggccacagcggccgcgctccggcctcgctccgccgctccacgcctcgcgggatccgcgggggcagcccggccgggcggggatgccggggctggggcggagggcgcagtggctgtgctggtggtgggggctgctgtgcagctgctgcgggcccccgccgctgcggccgcccttgcccgctgccgcggccgccgccgccggggggcagctgctgggggacggcgggagccccggccgcacggagcagccgccgccgtcgccgcagtcctcctcgggcttcctgtaccggcggctcaagacgcaggagaagcgggagatgcagaaggagatcttgtcggtgctggggctcccgcaccggccccggcccctgcacggcctccaacagccgcagcccccggcgctccggcagcaggaggagcagcagcagcagcagcagctgcctcgcggagagccccctcccgggcgactgaagtccgcgcccctcttcatgctggatctgtacaacgccctgtccgccgacaacgacgaggacggggcgtcggagggggagaggcagcagtcctggccccacgaagcagccagctcgtcccagcgtcggcagccgcccccgggcgccgcgcacccgctcaaccgcaagagccttctggcccccggatctggcagcggcggcgcgtccccactgaccagcgcgcaggacagcgccttcctcaacgacgcggacatggtcatgagctttgtgaacctggtggagtacgacaaggagttctcccctcgtcagcgacaccacaaagagttcaagttcaacttatcccagattcctgagggtgaggtggtgacggctgcagaattccgcatctacaaggactgtgttatggggagttttaaaaaccaaacttttcttatcagcatttatcaagtcttacaggagcatcagcacagagactctgacctgtttttgttggacacccgtgtagtatgggcctcagaagaaggctggctggaatttgacatcacggccactagcaatctgtgggttgtgactccacagcataacatggggcttcagctgagcgtggtgacaagggatggagtccacgtccacccccgagccgcaggcctggtgggcagagacggcccttacgacaagcagcccttcatggtggctttcttcaaagtgagtgaggtgcacgtgcgcaccaccaggtcagcctccagccggcgccgacaacagagtcgtaatcgctctacccagtcccaggacgtggcgcgggtctccagtgcttcagattacaacagcagtgaattgaaaacagcctgcaggaagcatgagctgtatgtgagtttccaagacctgggatggcaggactggatcattgcacccaagggctatgctgccaattactgtgatggagaatgctccttcccactcaacgcacacatgaatgcaaccaaccacgcgattgtgcagaccttggttcaccttatgaaccccgagtatgtccccaaaccgtgctgtgcgccaactaagctaaatgccatctcggttctttactttgatgacaactccaatgtcattctgaaaaaatacaggaatatggttgtaagagcttgtggatgccactaactcgaaaccagatgctggggacacacattctgccttggattcctagattacatctgccttaaaaaaacacggaagcacagttggaggtgggacgatgagactttgaaactatctcatgccagtgccttattacccaggaagattttaaaggacctcattaataatttgctcacttggtaaatgacgtgagtagttgttggtctgtagcaagctgagtttggatgtctgtagcataaggtctggtaactgcagaaacataaccgtgaagctcttcctaccctcctcccccaaaaacccaccaaaattagttttagctgtagatcaagctatttggggtgtttgttagtaaatagggaaaataatctcaaaggagttaaatgtattcttggctaaaggatcagctggttcagtactgtctatcaaaggtagattttacagagaacagaaatcggggaagtggggggaacgcctctgttcagttcattcccagaagtccacaggacgcacagcccaggccacagccagggctccacggggcgcccttgtctcagtcattgctgttgtatgttcgtgctggagttttgttggtgtgaaaatacacttatttcagccaaaacataccatttctacacctcaatcctccatttgctgtactctttgctagtaccaaaagtagactgattacactgaggtgaggctacaaggggtgtgtaaccgtgtaacacgtgaaggcaatgctcacctcttctttaccagaacggttctttgaccagcacattaacttctggactgccggctctagtaccttttcagtaaagtggttctctgcctttttactatacagcataccacgccacagggttagaaccaacgaagaaaataaaatgagggtgcccagcttataagaatggtgttagggggatgagcatgctgtttatgaacggaaatcatgatttcccttgtagaaagtgaggctcagattaaattttagaatattttctaaatgtctttttcacaatcatgtactgggaaggcaatttcatactaaactgattaaataatacatttataatctacaactgtttgcacttacagctttttttgtaaatataaactataatttattgtctattttatatctgttttgctgtaacattgaaggaaagaccagacttttaaaaaaaaagagtttatttagaaagtatcatagtgtaaacaaacaaattgtaccactttgattttcttggaatacaagactcgtgatgcaaagctgaagttgtgtgtacaagactcttgacagttgtgcttctctaggaggttgggtttttttaaaaaaagaattatctgtgaaccatacgtgattaataaagatttcctttaaggca 25 Rhesus BMP6 -agcgcagcccggactcggacgcacctggcctgtaccgcgcgcctctagagacctgcg XM_001085364.2cggggctgtggggctccccttcctcccctccaagcggttctcccggtgatcgccccttcgccacccctctatcctgggcaactgggggcgccccggacgaccatgagagataaggactgagggccaggaaggggaagcgagcccgccgagaggtggcgggggctgctcacgccaagggccacagcggccgtgctccagcctcgctccgccgctccacgcctcgcgggatccgcgggggcagcccggccgggcggggatgccggggctggggcggagggcgcagtggctgtgctggtggtgggggctgttgtgcagctgctgcgggcccccgccgctgcggccgcccctgcccgctgccgcggccgccgccgccgggggccagctgctgggggacggcgggagccccggccgcacggagcagccgccgccgtcgccgcaatcctcctcgggcttcctctaccggcggctcaagacgcacgagaagcgggagatgcagaaggagatcttgtcggtgctggggctcccacaccggccccggcccctgcacggcctccaacagccgcagcccccggcgctcccgcagcagcagcagcagcagcagcagccgcctcgcggagagccccctcccgggcagctgaagtccgcgcccctcttcatgctggatctgtacaacgccctgtccgccgacgacgaggaggacggggcgtcggagggggagaggcagcagccctggccccacgaaggagccagctcgtcccagcctcggcagccggccccgggcgccgcgcacccgctcaaccgcaagagcctcctggcccccggacctggcagcggcggcgcgtccccactgaccagcgcgcaggacagcgccttcctcaacgacgcagacatggtcatgagctttgtgaacctggtggagtacgacaaggagttctcccctcgtcagcgacaccacaaagagttcaagttcaacttatcccagattcctgagggtgaggcggtgacggctgcagaattccgcatctacaaggactgtgttgtggggagttttaaaaaccaaacttttcttatcagcatttatcaagtcttacaggagcatcagcacagagactctgacctttttttgctggacacccgcgtagtgtgggcctcagaagaaggctggctggaatttgacatcacggccactagcaatctgtgggttgtgaccccgcagcataacatggggcttcagctgagtgtggtgacgcgggatggagtccacatccatccccgagccgcgggcctggtgggcagagacggcccttacgacaagcagcccttcatggtggctttcttcaaagtgagtgaggtccacgtgcgcaccaccaggtcagcctctggccggcgccgacaacagagtcgtaatcgctctacccagtcccaggacgtggcgcgggtctccagtgcttcagattacaacagcagtgaattaaaaacagcttgcaggaagcatgagctgtatgtgagtttccaagacctgggatggcaggactggatcattgcacccaagggctacgctgccaattactgtgatggggaatgctccttcccactcaacgcacacatgaatgcaaccaaccacgcgatcgtgcagaccttggttcaccttatgaaccctgagtatgtccccaaaccgtgctgtgcgccaactaaactaaatgccatctcagttctttactttgatgacaattccaatgtcattctgaaaaaatacaggaatatggttgtaagagcttgtggatgccactaactcgaaaccagatgctggggacacacattctgccttggattcctagattacatctgccttaaaaaacacagaagcacagttggaggtgggacgatgagacttggaaactatctcatgccagtgccttattacccaagaagattttaaaggacctcattaataatttgctcacttggtaaatgacgtgagtagttgttggtctgtagcaagctgagtttggatgtctgtagtgcaaggtccggtaactgcagaaagcaccgtgaagctcttcctcccctcctcccccaaaaacccaccaaaattagttttagctgtagatcaagctatttggggtgttagtaagtagggaaaataatctcaaaggagttaaatgtattcttggttaaagtatcagcctgttcagtactgtctatcaaaggtagattttacagagaacagaaattggggaagttgggggaacgcctctgttcagatttcattcccaggaagttcaatttcatacatgacccacagcccaggccacagccagggctccatggggcgcctttgtctcagtcattgctgttatgtgttcatgctggagttttgttg 26 Mouse BMP6 -gatcctggccgtcgccccgtcgtctcttctccacccgggcttctgggggcgccgcgg NM_007556.2atgaccatgagagataaggactgagtgccaggaccgggaagagagcccgccgagaggtggcgggggctgcccactccgagggccacagcctccgcgctccggcctcgctccgccgctcgacgcctcgcgggccccgcgggggcagccgggctgggcggcgatgcccgggctggggcggagggcgcagtggctgtgctggtggtgggggttgctgtgcagctgcggccccccgccactgcggccccctctgccggtagccgcggccgccgccggggggcagctgctgggagccggcgggagcccggtgcgcgctgagcagccaccgccacagtcctcttcttcgggcttcctctatcggcggctcaagacccacgagaagcgggagatgcaaaaggagatcctgtcggtgctggggctcccgcacaggccgcggcccctgcacggtctccagcagcctcagcccccggtgctcccgccacagcagcagcagcagcagcagcagcagcagacggcccgcgaggagccccctccagggcggctgaagtccgctccactcttcatgctggatctctacaacgccctgtccaatgacgacgaagaggatggggcatcggagggtgtggggcaagagcctgggtcccacggaggggccagctcgtcccagctcaggcagccgtctcccggcgctgcacactccttgaaccgcaagagtctcctggccccgggacccggtggcggtgcgtccccactgactagcgcgcaggacagcgctttcctcaacgacgcggacatggtcatgagctttgtgaacctggtggagtacgacaaggagttctccccacatcaacgacaccacaaagagttcaagttcaacctatcccagattcctgagggtgaggcggtgacggctgctgagttccgcgtctacaaggactgtgtggtggggagttttaaaaaccaaacctttcttatcagcatttaccaagtcttgcaggagcatcagcacagagactctgacctatttttgttggacacccgggtggtgtgggcctcagaagaaggttggctggaatttgacatcacagcaactagcaatctgtgggtggtgacaccgcagcacaacatggggctccagctgagtgtggtgactcgggatggactccacgtcaacccccgtgcggcgggcctggtgggcagagacggcccttacgacaagcagcccttcatggtggccttcttcaaggtgagcgaggtccacgtgcgcaccaccaggtcagcctccagtcggcggcggcagcagagtcgcaaccggtccacccagtcgcaggacgtgtcccggggctccggttcttcagactacaacggcagtgagttaaaaacagcttgcaagaagcatgagctctatgtgagcttccaggacctgggatggcaggactggatcattgcacccaaaggctacgctgccaactactgtgatggagagtgttccttcccactcaacgcacacatgaatgccaccaaccacgccattgtacagaccttggtccaccttatgaatcccgagtacgtccccaaaccatgctgcgcaccaaccaaactgaatgccatctcggttctttacttcgatgataactccaatgtcatcttgaaaaagtacaggaatatggtcgtgagagcttgtggttgccattaagttgaagctggtgtgtgtgtgtgggtgggggcatggttctgccttggattcctaacaacaacatctgccttaaaccacgaacaacagcacagcgaagcgggatggtgacacacagagggatcgtgacacgcagacacatctcccgctggtgccttacccacggaggcttttatgaggaccttgtcaagggctttcccagttcctaactgagcagttgctggtctgcaggaagctggaaggcttgtagtacaggcctggaaactgcagttacctaatgttcgcctcccccaaccccgcccggagtagttttagcttttagatctagctgcttgtggtgtaagtagagagtaaacttgaaggaatattaaatatccctgggttgaaagacccggtggtggctctacagcacccatcccagggagatttttgcagacatccgaatggaggggagaagggcactctttcaggttccattcccagcaagggcagctcacacaggacctgcagcctggccatcagcaggctctgtggaggtgccttctgtctactgttgtagttacgtgttttgtgttgactctcggtggtgtgagaatgtactaatctctgtcaagacaaactgtagcatttccaccccatcctcctccctccctcacagaattc 27 Rat BMP6 -atgcccgggctggggcggagggcgcagtggctgtgctggtggtgggggttactgtgc NM_013107.1agctgcggccccccgccactgcggccccctctgccggtagccgcggccgccgccggggggcagctgctgggagccggcgggagccctgtgcgcgccgagcagccaccgccgcaatcctcctcttcgggcttcctctatcggcggctcaagacccacgagaagcgggagatgcaaaaggagatcctgtcggtgttggggctgcctcacaggccgcggcccctgcacggtctccagcagcctcaatcccccgtgctcccgcagcagcaacaatcgcaacagacggcccgcgaggagccccctccagggcggctgaagtccgctccgctcttcatgctggatctctacaactccctgtccaaggacgacgaagaggatggggtgtcagagggagagggactggagcccgagtcccacggaagggccagctcgtcccagctcaaacagccatctcccggggctgcacactccctgaaccgcaagagtctcctggccccgggacccggcggcagtgcgtccccactgaccagcgcgcaggacagcgctttcctcaacgacgcggacatggtcatgagctttgtgaacctggtggagtacgacaaggagttctccccacgccagcgacaccacaaggagttcaagttcaacttatcccagattcccgagggtgaggcagtgacggctgcagagttccgcgtctacaaggactgtgtggtggggagttttaaaaaccaaacttttcttatcagcatttaccaagtcttacaggagcatcagcacagagactctgacctatttttgttggacacccgggtggtgtgggcctccgaagaaggctggctggaattcgacatcacagcaactagcaatctgtgggtggtgacaccgcagcacaacatgggactccagctgagtgtggtgactcgggacggactccacatcaacccccgtgcggcgggcctggtgggcagagacggcccttacgacaagcagcccttcatggtggccttcttcaaggtgagcgaggtccacgtgcgcaccaccaggtcagcctccagtcggcgtcgacagcagagtcgcaatcggtccacccagtcgcaggacgtgtcccggggctccagtgcttcagactacaacagcagtgagttaaaaacagcttgcaagaagcatgagctttacgtgagcttccaggacctgggatggcaggactggatcatcgcacccaaaggctacgctgccaactattgtgacggagagtgttccttccctctcaatgcacacatgaatgccaccaaccacgccattgtacagaccttggtccaccttatgaatcccgagtacgtccccaaaccatgctgcgcaccaaccaaactgaatgccatctcggttctttacttcgacgacaactccaatgtcatcttgaaaaaatacaggaacatggttgtgagagcttgtggatgtcattga 28 Human NEO1 -gggccgggccgggctgggctggagcagcggcggccgcgggagccgagcttgcagcga NM_002499.2gggaccggctgaggcgcgcgggagggaaggaggcaagggctccgcggcgctgtcgccgccgctgccgctcactctcggggaagagatggcggcggagcggggagcccggcgactcctcagcaccccctccttctggctctactgcctgctgctgctcgggcgccgggcgccgggcgccgcggccgccaggagcggctccgcgccgcagtccccaggagccagcattcgaacgttcactccattttattttctggtggagccggtggatacactctcagttagaggctcttctgttatattaaactgttcagcatattctgagccttctccaaaaattgaatggaaaaaagatggaacttttttaaacttagtatcagatgatcgacgccagcttctcccggatggatctttatttatcagcaatgtggtgcattccaaacacaataaacctgatgaaggttattatcagtgtgtggccactgttgagagtcttggaactattatcagtagaacagcgaagctcatagtagcaggtcttccaagatttaccagccaaccagaaccttcctcagtttatgctgggaacaatgcaattctgaattgtgaagttaatgcagatttggtcccatttgtgaggtgggaacagaacagacaaccccttcttctggatgatagagttatcaaacttccaagtggaatgctggttatcagcaatgcaactgaaggagatggcgggctttatcgctgcgtagtggaaagtggtgggccaccaaagtatagtgatgaagttgaattgaaggttcttccagatcctgaggtgatatcagacttggtatttttgaaacagccttctcccttagtcagagtcattggtcaggatgtagtgttgccatgtgttgcttcaggacttcctactccaaccattaaatggatgaaaaatgaggaggcacttgacacagaaagctctgaaagattggtattgctggcaggtggtagcctggagatcagtgatgttactgaggatgatgctgggacttatttttgtatagctgataatggaaatgagacaattgaagctcaagcagagcttacagtgcaagctcaacctgaattcctgaagcagcctactaatatatatgctcacgaatctatggatattgtatttgaatgtgaagtgactggaaaaccaactccaactgtgaagtgggtcaaaaatggggatatggttatcccaagtgattattttaagattgtaaaggaacataatcttcaagttttgggtctggtgaaatcagatgaagggttctatcagtgcattgctgaaaatgatgttggaaatgcacaagctggagcccaactgataatccttgaacatgcaccagccacaacgggaccactgccttcagctcctcgggatgtcgtggcctccctggtctctacccgcttcatcaaattgacgtggcggacacctgcatcagatcctcacggagacaaccttacctactctgtgttctacaccaaggaagggattgctagggaacgtgttgagaataccagtcacccaggagagatgcaagtaaccattcaaaacctaatgccagcgaccgtgtacatctttagagttatggctcaaaataagcatggctcaggagagagttcagctccactgcgagtagaaacacaacctgaggttcagctccctggcccagcacctaaccttcgtgcatatgcagcttcgcctacctccatcactgttacgtgggaaacaccagtgtctggcaatggggaaattcagaattataaattgtactacatggaaaaggggactgataaagaacaggatgttgatgtttcaagtcactcttacaccattaatgggttgaaaaaatatacagagtatagtttccgagtggtggcctacaataaacatggtcctggagtttccacaccagatgttgctgttcgaacattgtcagatgttcccagtgctgctcctcagaatctgtccttggaagtgagaaattcaaagagtattatgattcactggcagccacctgctccagccacacaaaatgggcagattactggctacaagattcgctaccgaaaggcctcccgaaagagtgatgtcactgagaccttggtaagcgggacacagctgtctcagctgattgaaggtcttgatcgggggactgagtataatttccgagtggctgctctaacaatcaatggtacaggcccggcaactgactggctgtctgctgaaacttttgaaagtgacctagatgaaactcgtgttcctgaagtgcctagctctcttcacgtacgcccgctcgttactagcatcgtagtgagctggactcctccagagaatcagaacattgtggtcagaggttacgccattggttatggcattggcagccctcatgcccagaccatcaaagtggactataaacagcgctattacaccattgaaaatctggatcccagctctcactatgtgattaccctgaaagcatttaataacgtgggtgaaggcatccccctgtatgagagtgctgtgaccaggcctcacacagacacttctgaagttgatttatttgttattaatgctccatacactccagtgccagatcccactcccatgatgccaccagtgggagttcaggcttccattctgagtcatgacaccatcaggattacgtgggcagacaactcgctgcccaagcaccagaagattacagactcccgatactacaccgtccgatggaaaaccaacatcccagcaaacaccaagtacaagaatgcaaatgcaaccactttgagttatttggtgactggtttaaagccgaatacactctatgaattctctgtgatggtgaccaaaggtcgaagatcaagtacatggagtatgacagcccatgggaccacctttgaattagttccgacttctccacccaaggatgtgactgttgtgagtaaagaggggaaacctaagaccataattgtgaattggcagcctccctccgaagccaatggcaaaattacaggttacatcatatattacagtacagatgtgaatgcagagatacatgactgggttattgagcctgttgtgggaaacagactgactcaccagatacaagagttaactcttgacacaccatactacttcaaaatccaggcacggaactcaaagggcatgggacccatgtctgaagctgtccaattcagaacacctaaagcggactcctctgataaaatgcctaatgatcaagcctcagggtctggagggaaaggaagccggctgccagacctaggatccgactacaaacctccaatgagcggcagtaacagccctcatgggagccccacctctcctctggacagtaatatgctgctggtcataattgtttctgttggcgtcatcaccatcgtggtggttgtgattatcgctgtcttttgtacccgtcgtaccacctctcaccagaaaaagaaacgagctgcctgcaaatcagtgaatggctctcataagtacaaagggaattccaaagatgtgaaacctccagatctctggatccatcatgagagactggagctgaaacccattgataagtctccagacccaaaccccatcatgactgatactccaattcctcgcaactctcaagatatcacaccagttgacaactccatggacagcaatatccatcaaaggcgaaattcatacagagggcatgagtcagaggacagcatgtctacactggctggaaggcgaggaatgagaccaaaaatgatgatgccctttgactcccagccaccccagcctgtgattagtgcccatcccatccattccctcgataaccctcaccatcatttccactccagcagcctcgcttctccagctcgcagtcatctctaccacccgggcagcccatggcccattggcacatccatgtccctttcagacagggccaattccacagaatccgttcgaaatacccccagcactgacaccatgccagcctcttcgtctcaaacatgctgcactgatcaccaggaccctgaaggtgctaccagctcctcttacttggccagctcccaagaggaagattcaggccagagtcttcccactgcccatgttcgcccttcccacccattgaagagcttcgccgtgccagcaatcccgcctccaggacctcccacctatgatcctgcattgccaagcacaccattactgtcccagcaagctctgaaccatcacattcactcagtgaagacagcctccatcgggactctaggaaggagccggcctcctatgccagtggttgttcccagtgcccctgaagtgcaggagaccacaaggatgttggaagactccgagagtagctatgaaccagatgagctgaccaaagagatggcccacctggaaggactaatgaaggacctaaacgctatcacaacagcatgacgaccttcaccaggacctgacttcaaacctgagtctggaagtcttggaacttacccttgaaaacaaggaattgtacagagtacgagaggacagcacttgagaacacagaatgagccagcagactggccagcgcctctgtgtagggctggctccaggcatggccacctgccttcccctggtcagcctggaagaagcctgtgtcgaggcagcttccctttgcctgctgatattctgcaggactgggcaccatgggccaaaattttgtgtccagggaagaggcgagaagtgcaacctgcatttcactttgtggtcaggccgtgtctttgtgctgtgactgcatcacctttatggagtgtagacattggcatttatgtacaattttatttgtgtcttattttattttaccttcaaaaacaaaaacgccatccaaaaccaaggaagtccttggtgttctccacaagtggttgacatttgactgcttgttccaattatgtatggaaagtctttgacagtgtgggtcgttcctggggttggcttgttttttggtttcatttttattttttaattctgagtcattgcatcctctaccagctgttaatccatcactctgagggggaggaaatgttgcattgctgtttgtaagctttttttattatttttttattataattattaaaggcctgactctttcctctcatcactgtgagattacagatctatttgaattgaatgaaatgtaacattgaaaagacttgtttgttgctttctgtgcagtttcagtattggggcgggtggggggctgggggttggtaataggaaatggaggggctgctgaggtcctgtgaatgtttctgtcattgtactttcttccagaagcctgcagagaatggaagcatcttctttattgtcctttcctggcatgtccatccttattgtcactacgttgcaactggagtttgatttggatctggttttaaaattcttctgtgcaatagatgggtttgaggatttagcggccctgatgtcttggtcatagcctggtaagaatgtccatgctgaggagccagatgttgtatttctaactgcctgagtcacacagaatagggtaagagcctgaccccattctgtaaatcagaaagcaaggatggagaccctttcctgctgctattattggctctctttgaggaagttggaggttaaggaaggaacttgtttgtttccgtatacgactccttcttctctctagttcagtcttcagccagtccagcgctctcttccacacttcagagccccttcagagaaagcattagcaggaatgagacaaggcagagctgcagtgccccctgaggcttccacacatctttctgaatattatttttcaagtaacaagggcagggacagcggaaacagctgcccaccccccccatcccagcagctcagctaagccctgatgagaatgaagccacaggagttgtctgaggtgaacccagccgctcagccacacatggaagccattgcctttgcacatagttcttgggttctttttcctaaaaaggtaaggagctgaggtgtgtggttttttaatattaagaatatataatggaaaacacacgactgacgctcaggcatcttcccctactccccaacagatccccagaagacagcgtggaaggcagtgtagacagtaaatcgggcttcagttctatagccaagaagagatcagctgctgaaaccaccagtgggtaccccaggccacctgcctttgaacttggggatttgccatgtttgatcttgtcacatacttgcttttttacaagatgaactctttgtatttatgatttggggggcaatgaaaggtgcaatgcaggaactgctgctgccgagctcgctggtcacatgggggtgccaggcgggattctggaaaaccagtgcacttaaactgatcctgaagagagctgtcccagcactctggccaccaggagggccagattccccagaaactaccttttgcccaaagaacatgctcagtatttggggcatttcctcccacaaaccctgactgcttctgttacctcagggccttggtacctggatactgccacagaattggggcgggtgggggaggggcctatttttaaataaaataactgttcaaagttgggggttttttaaaaaattaagaaaaaggaaagctattctgtattgcaccttttcacaatttaatacattttcttacattttcctgtgattttcgaaactaaaccattgtgtgtcctgtagtgtcctggttgagctgccgctcagcagcttcctcggggggatttggaacacctgtgtctgtcgccgcactgcctgtgggaggggcccagagggctgctgggactggcgtctgtacacacttgtttggccttttctgtagttgatgctgtaaactctatggctttttaaaaacgatttcatgtttttatttagtattggaaatccaatacacttttttaatccaatcaaaaaaaaaaaaaaaaaaaaaaa 29 Mouse NEO1 -gcccccctcgctctaccgtgaggagcccgagtcggcggcgggtggcggcgcctggaa NM_001042752.1cctggagagaccgagccaccccccggctctcggccggaatgtactgattctcctctgctctcctccccgccccgctgcaggagggaggcgcccggagtctttccccctgggcgcgcgagggggccgcgcgggccgggccgggccgggctggagccgagccctgcggcgcagagaccggctgaggcgcgctgagggaagggcgcgagcgctccgcggcgctatcgccgccgccgccgccgccactcgtggggtagagatggcggcggagcgcgaagccgggcgactcctctgcacctcctcctcccggcgctgctgtccgccaccgccgctgctgctgttgctgccgctgctgctgctgctcggacgcccggcgtccggcgccgcggccacgaagagcggctccccgccgcagtccgcaggagccagtgttcgaacattcactccgttttattttctggtggagccagtagacaccctctcagttagaggctcttctgttatattaaattgctcggcatattctgagccctctccaaacattgaatggaagaaagatgggacttttttaaacttagaatcagatgatcgacgccagctactcccagatggatctttattcatcagcaacgtggtgcattccaaacacaataagcctgacgaaggtttctatcagtgtgtagccactgtggataatcttggaaccattgtcagcagaacagccaagctcacagtagcaggtcttccaagatttaccagccaaccagaaccttcttcagtctatgttggaaacagtgcaattctgaattgtgaagttaatgcagatttggtcccatttgttaggtgggaacagaatcgacagccccttcttctagatgacaggattgtcaaacttccaagtggaacactggttatcagcaatgctactgaaggagatgggggactctaccgctgcattgttgaaagtggtgggccaccaaagtttagtgacgaagctgaattgaaagttcttcaagatcctgaggaaattgtagacttggtatttctgatgcgaccatcttctatgatgaaagtcactggtcagagtgcagtgttgccatgtgttgtctcagggcttcctgctccagttgttagatggatgaaaaacgaagaagtgcttgacacagaaagctctggcaggttggtcttgctagcaggaggttgcttggagatcagtgatgtcactgaggatgatgctgggacttatttttgcatagctgataatggaaataagacagttgaagctcaggcggagcttactgtgcaagtgccacctggattcctgaaacaacctgctaacatatatgctcacgaatccatggacattgtatttgaatgtgaagtcactgggaagccaactccaactgtgaagtgggtcaagaatggggatgtggttatccccagtgattactttaaaattgtaaaggaacataatcttcaagttttgggtctggtgaaatcagatgaagggttctatcaatgcattgctgagaatgatgttggaaatgcacaagctggagcccagctgataatccttgagcatgatgttgccatcccaacattacctcccacttcactgaccagtgccactactgaccatctagcaccagccacaacgggaccattaccttcagctcctcgagacgtcgtggcctccctggtctctactcgcttcattaaattgacatggcgtacacctgcatcagaccctcatggagacaatctcacctactctgtgttctacaccaaggaaggggttgctagggagcgtgttgagaataccagccagccaggagagatgcaggtgactattcaaaacttgatgccagcaactgtgtacatcttcaaagttatggctcaaaataagcatggctctggagaaagttcagctcctcttcgagtagagacacagcctgaggttcagctccctggcccagcacctaatatccgtgcttatgcaacgtcacctacttctatcactgtcacctgggaaacaccgttatctggcaatggggaaattcaaaattacaaattgtactacatggaaaaaggaactgataaagaacaggatattgatgtttcaagtcactcctacaccattaatggactgaagaaatacacagaatacagtttccgagtggtggcctacaataaacatggtcctggagtttctacacaagatgttgctgttcgaacattatcagatgttcccagtgctgctcctcagaatctgtccttagaagtgagaaattcaaagagtatagtgatccactggcagcccccttcctcaaccacacaaaatgggcagataactggctacaagattcgatatcgaaaggcctcccgaaaaagtgatgtcactgagaccttggtaactgggacacagctgtctcagctgattgaaggtcttgatcgggggacagaatataacttccgagtcgctgctctcacagtcaatggtacaggtccagcaactgattggctgtctgctgaaacttttgaaagcgacctagatgaaactcgtgttcctgaagtgcccagctctcttcatgtccgtccgctcgtcactagcattgtagtgagctggactcctccagagaaccagaacattgtggtccgaggttatgccatcggttacggcattggcagccctcatgcccagaccatcaaagtggactataaacaacgttattacaccatcgaaaacttggatccaagctctcattacgtgattaccttgaaagcatttaacaatgttggcgaaggcatccccctttatgagagtgctgtgaccagacctcacacagtgccagatcccactcccatgatgccaccagtgggagttcaggcttccattctgagtcacgacaccataaggattacctgggcagacaactccctgcccaaacaccagaagattacagactcccgctactacacagtccggtggaagaccaacatcccagcaaacacgaagtacaagaatgcaaatgcaacgacgttaagctatttggttactggtttaaagccaaatacgctctatgagttctctgtgatggtgaccaaaggcagaaggtcaagcacgtggagtatgacagctcatggcgctacctttgaattagttcctacttctccacctaaggatgtgacagttgtgagtaaggaaggaaaacctagaaccatcatagtgaattggcagcctccctctgaagctaacggcaagattacaggttacatcatctattacagcacggatgtgaatgcagagatacatgactgggttattgaaccagttgtgggaaacagactgactcaccagattcaagagttaacacttgatacgccatactacttcaaaatccaggcccggaactcaaagggcatggggcccatgtctgaagctgtacagttcagaacacctaaagccttagggtcagcaggaaaaggaagccgactaccagacctgggatctgactacaaacctccaatgagtggcagcaacagccctcacgggagccccacctcccctctggacagcaacatgctgctggtcatcattgtctctgttggcgtcatcactatcgtggtggttgtggtcattgctgtcttttgtacccggcgcaccacctctcaccagaagaagaaacgagctgcgtgcaaatcagtgaatggctcccataagtacaagggcaattgcaaagatgtgaagcctccagacctatggatccatcacgagagactagagttgaagcctattgacaagtctccagatcctaaccctgtcatgactgatactccaatccctcgaaactctcaagatatcacaccagtggacaattccatggatagcaatatccatcaaaggcggaattcatacagagggcatgagtcagaggacagcatgtctacactggctggaaggaggggaatgagaccaaaaatgatgatgccctttgactctcagccacctcagcctgtgattagtgcccatcccatccattccctcgataaccctcaccatcatttccactccagcagcctcgcttctccagcccgcagtcatctctaccacccaagcagcccatggcccattggcacatccatgtccctttcagacagggccaattccacagaatctgttcgaaatacccccagcacggacaccatgccagcgtcctcgtctcagacgtgctgcactgaccatcaggaccctgagggtgctactagctcctcttacttggccagctcccaagaggaagactcaggccagagtcttcccacagcccatgtccgcccttcccaccctctgaagagcttcgctgtgccagcaatcccacccccaggacctcctctctatgatcctgcactgccaagcacaccattactgtcccagcaagctctgaaccatcacattcactcagtgaaaacagcctccatcgggacgttaggaaggagccggcctcctatgccagtggttgttccgagtgcccctgaagtacaggagaccaccaggatgctggaagactccgagagtagctatgaaccagatgagctgaccaaagagatggcccacctggaaggactaatgaaggacctaaatgccatcacaacagcctgatgaccttcgcctggacatgactccaagcctgagtctacaagtctcggaacttaaccttgaaaacaaggaattgtacagagtacgagaggacagcacttgagagcaggagccagcaaaccagccagtgcctccatgtggggttggctccaggcacagccacctgccttctcctggtcagcctggattacacttgtgtggaggcagcttccctttgcctgctgagagcctgcaggactgggcactatgggccaaaattttgtgtccagggaagaggcaagaagtacgacctgccttttgctttgtggtcagtggcttgtgtctttgtgctgcaactgcatcacttttatggagtgtagacattggcatttatgtacaattttgtgtcctattttattttaccttaaaacactatcagaagccaagggagtctgtgatgttctctcaagcagttgacacttgactgtggttccagttacttacggaaagtcatcaacagtgaggttgtttgacaccactgacaggcattggcttgttgtgggtttcatttttattcttaattctgagacattgcatcctctgccagctgttaatccatcactttgaggggaggacatgttgcattgctgtttgtaagcttttttattatttttttattataattattaaaggcctgactttctcctctcatcactgtgagattacagatctatttgaatgaaatgtaacattgaaaagacttgtttgttgctttctgtgcagattcagtattggggtgggattggggattgggaataggaaatggaggggctgctgaggccctgtgaatgtttctgtcaatgtactttgttccagaagcctgccgagaatgaaagcagcatctttagtgtcctttcctggcatttccatcttcgtgtcactgcatagcaactggagttttgtttggatctggttataaattcttgtacagtggatgactttggtgatttagctgccctggtatcttggtcatttcctctttggagtgtccacactgaggtctctatcaatgtatgtttaattgcttgagagatgccaagtagaaccagagcctgactgtgctctgagaagctacaaagcacagggtggagactccctttgtgttgctagtattggttctctctggaaggttaaaatctaaggcaggatcttggtttcctattccaaataggatgcctgcttctctgggcaccagtcctcagccaggcagctctcgtggcattgcagaggctctcctgaaaaacatcaaccagggtgagagccaagatggggtggcacccatgacgcttccccacatgtttcttcaaggagcagaggacagagatagtggaaagagggtcagcagaagcaggtgccttcatctatcccagcagctcagccaaaccccagttagaatgaggcagcaggagattccaggtgtgctgagggttcagccacacgcagaagacgttgcagagtgttaaagaggtaagctgaggtgtgtatttggttggctttgttgttgttgttaatgtataatgaaaagtataagactaaccctcaggcctcatgttctccaatagatccctggaagacagtatagaaagtcagtcgggcttgggctccttagccagtgagactactcagaccaccagtggctagcctagcctacctgtccttgaacatgggtgattttacccctttgaggtcttaaccctttttttactttcaacaagatgagctctttgtatgattgcgggcgggggatatgaaaatgcaatgatctaactcctgttgctcttctagctggtcacatgacggcaccaggcagggttctgggacacccggtgtgctttgactgttctacaaaaagctgtcagagcgttctggcctcctggaggctagattcctcagaaactgtctagcctttgcccacagagcatgctatgtaattagagcactccttcccatgaaccccagcacttgtgttacctcagggccttggtacctggatactgccacagaatttccatggggcgggaagggatgtatttttaaataaagtaacttaaaagttggggaaattttttaaattcagaaaatgcaaagctattctgtattacaccatttcacaatttaatatgtcttatattttcctgtgactctggaaactaaaccattgtgtgtcttgtcgtgtcctagttgagctggggcctagcagcttccttccagtgggtgtggagcaaacgtgtatgtcgcctcgctacctgcttgaggggtccgaagggctgctgggactgagttctgtacacacttgtttggccttttctgtagttgatgctgtaaaactctatggctttttaaaaacaatttcatgtttttattttgtattggaagtccaatacacttttttaatccaatcaaactggtctggtcaaaaagttctttcccttaaaagttcaggggctcctacttccagcttccgatgacttctctgtggctctcactgctataaagcaggatttagaatggcaatctgggcagaggtaataaaagaaatgtctgactgc cagccccaaaa

TABLE 2 siRNA targeting HAMP 3′ UTR SEQ SEQ Duplex ID Sense ID Antisensename Start NO (5′-3′) NO (5′-3′) 307-325_s 307 53 GGAUGUGCUGCAAGACGUA 96UACGUCUUGCAGCACAU CC 309-327_s 309 54 AUGUGCUGCAAGACGUAGA 97UCUACGUCUUGCAGCAC AU 310-328_s 310 55 UGUGCUGCAAGACGUAGAA 98UUCUACGUCUUGCAGCA CA 313-331_s 313 56 GCUGCAAGACGUAGAACCU 99AGGUUCUACGUCUUGCA GC AD- 314 57 CUGCAAGACGUAGAACCUA 100UAGGUUCUACGUCUUGC 11439.1_314-332_s AG 322-340_s 322 58CGUAGAACCUACCUGCCCU 101 AGGGCAGGUAGGUUCUA CG 347-365_s_G1A 347 59GUCCCCUCCCUUCCUUAUU 102 AAUAAGGAAGGGAGGGG AC 348-366_s 348 60UCCCCUCCCUUCCUUAUUU 103 AAAUAAGGAAGGGAGGG GA 349-367_s 349 61CCCCUCCCUUCCUUAUUUA 104 UAAAUAAGGAAGGGAGG GG 350-368_s 350 62CCCUCCCUUCCUUAUUUAU 105 AUAAAUAAGGAAGGGAG GG 351-369_s 351 63CCUCCCUUCCUUAUUUAUU 106 AAUAAAUAAGGAAGGGA GG 352-370_s_C19A 352 64CUCCCUUCCUUAUUUAUUA 107 UAAUAAAUAAGGAAGGG AG 352-370_s_C19U 352 65CUCCCUUCCUUAUUUAUUU 108 AAAUAAAUAAGGAAGGG AG 354-372_s 354 66CCCUUCCUUAUUUAUUCCU 109 AGGAAUAAAUAAGGAAG GG 355-373_s_G19A 355 67CCUUCCUUAUUUAUUCCUA 110 UAGGAAUAAAUAAGGAA GG 355-373_s_G19U 355 68CCUUCCUUAUUUAUUCCUU 111 AAGGAAUAAAUAAGGAA GG 356-374_s_C19A 356 69CUUCCUUAUUUAUUCCUGA 112 UCAGGAAUAAAUAAGGA AG 356-374_s_C19U 356 70CUUCCUUAUUUAUUCCUGU 113 ACAGGAAUAAAUAAGGA AG 357-375_s 357 71UUCCUUAUUUAUUCCUGCU 114 AGCAGGAAUAAAUAAGG AA 358-376_s_G19A 358 72UCCUUAUUUAUUCCUGCUA 115 UAGCAGGAAUAAAUAAG GA 358-376_s_G19U 358 73UCCUUAUUUAUUCCUGCUU 116 AAGCAGGAAUAAAUAAG GA 359-377_s_C19A 359 74CCUUAUUUAUUCCUGCUGA 117 UCAGCAGGAAUAAAUAA GG 359-377_s_C19U 359 75CCUUAUUUAUUCCUGCUGU 118 ACAGCAGGAAUAAAUAA GG 363-381_s 363 76AUUUAUUCCUGCUGCCCCA 119 UGGGGCAGCAGGAAUAA AU 365-383_s 365 77UUAUUCCUGCUGCCCCAGA 120 UCUGGGGCAGCAGGAAU AA 366-384_s 366 78UAUUCCUGCUGCCCCAGAA 121 UUCUGGGGCAGCAGGAA UA 369-387_s 369 79UCCUGCUGCCCCAGAACAU 122 AUGUUCUGGGGCAGCAG GA 370-388_s 370 80CCUGCUGCCCCAGAACAUA 123 UAUGUUCUGGGGCAGCA GG 373-391_s 373 81GCUGCCCCAGAACAUAGGU 124 ACCUAUGUUCUGGGGCA GC 375-393_s 375 82UGCCCCAGAACAUAGGUCU 125 AGACCUAUGUUCUGGGG CA 376-394_s 376 83GCCCCAGAACAUAGGUCUU 126 AAGACCUAUGUUCUGGG GC 379-397_s 379 84CCAGAACAUAGGUCUUGGA 127 UCCAAGACCUAUGUUCU GG 380-398_s 380 85CAGAACAUAGGUCUUGGAA 128 UUCCAAGACCUAUGUUC UG 381-399_s 381 86AGAACAUAGGUCUUGGAAU 129 AUUCCAAGACCUAUGUU CU AD- 382 87GAACAUAGGUCUUGGAAUA 130 UAUUCCAAGACCUAUGU 11442.1_382-400_s UC 383-401_s383 88 AACAUAGGUCUUGGAAUAA 131 UUAUUCCAAGACCUAUG UU 396-414_s 396 89GAAUAAAAUGGCUGGUUCU 132 AGAACCAGCCAUUUUAU UC 398-416_s 398 90AUAAAAUGGCUGGUUCUUU 133 AAAGAACCAGCCAUUUU AU 399-417_s 399 91UAAAAUGGCUGGUUCUUUU 134 AAAAGAACCAGCCAUUU UA 402-420_s 402 92AAUGGCUGGUUCUUUUGUU 135 AACAAAAGAACCAGCCA UU 403-421_s 403 93AUGGCUGGUUCUUUUGUUU 136 AAACAAAAGAACCAGCC AU 407-425_s 407 94CUGGUUCUUUUGUUUUCCA 137 UGGAAAACAAAAGAACC AG AD- 291 95CAUCGAUCAAAGUGUGGGA 138 UCCCACACUUUGAUCGA 11436.1_291-309_s UG Note thatan overhang (e.g. TT, dTsdT) can be added to the 3′ end of any duplex.

TABLE 3 siRNA targeting HAMP CDS SEQ SEQ Duplex ID Sense ID Antisensename Start NO (5′-3′) NO (5′-3′) 62-80_s_G19U 62 139 AGACGGCACGAUGGCA186 AAGUGCCAUCGUGC CUU CGUCU 67-85_s_C19A 67 140 GCACGAUGGCACUGAG 187UAGCUCAGUGCCAU CUA CGUGC 67-85_s_C19U 67 141 GCACGAUGGCACUGAG 188AAGCUCAGUGCCAU CUU CGUGC 74-92_s_C19A 74 142 GGCACUGAGCUCCCAG 189UAUCUGGGAGCUCA AUA GUGCC 74-92_s_C19U 74 143 GGCACUGAGCUCCCAG 190AAUCUGGGAGCUCA AUU GUGCC 76-94_s_G19A 76 144 CACUGAGCUCCCAGAU 191UAGAUCUGGGAGCU CUA CAGUG 76-94_s_G19U 76 145 CACUGAGCUCCCAGAU 192AAGAUCUGGGAGCU CUU CAGUG 132-150_s 132 146 CUGACCAGUGGCUCUG 193AAACAGAGCCACUG UUU GUCAG 140-158_s 140 147 UGGCUCUGUUUUCCCA 194UUGUGGGAAAACAG CAA AGCCA 146-164_s_hcU1C_G19A 146 148 UGUUUUCCCACAACAG195 UGUCUGUUGUGGGA ACA AAACA 146-164_s_hcU1C_G19U 146 149UGUUUUCCCACAACAG 196 AGUCUGUUGUGGGA ACU AAACA 155-173_s 155 150ACAACAGACGGGACAA 197 AAGUUGUCCCGUCU CUU GUUGU 157-175_s_C19A 157 151AACAGACGGGACAACU 198 UCAAGUUGUCCCGU UGA CUGUU 157-175_s_C19U 157 152AACAGACGGGACAACU 199 ACAAGUUGUCCCGU UGU CUGUU 160-178_s 160 153AGACGGGACAACUUGC 200 UCUGCAAGUUGUCC AGA CGUCU 161-179_s_G19A 161 154GACGGGACAACUUGCA 201 UUCUGCAAGUUGUC GAA CCGUC 161-179_s_G19U 161 155GACGGGACAACUUGCA 202 AUCUGCAAGUUGUC GAU CCGUC 162-180_s_C19A 162 156ACGGGACAACUUGCAG 203 UCUCUGCAAGUUGU AGA CCCGU 162-180_s_C19U 162 157ACGGGACAACUUGCAG 204 ACUCUGCAAGUUGU AGU CCCGU 242-260_s_C19A 242 158GAGGCGAGACACCCAC 205 UAAGUGGGUGUCUC UUA GCCUC 242-260_s_C19U 242 159GAGGCGAGACACCCAC 206 AAAGUGGGUGUCUC UUU GCCUC 253-271_s 253 160CCCACUUCCCCAUCUG 207 AUGCAGAUGGGGAA CAU GUGGG 258-276_s 258 161UUCCCCAUCUGCAUUU 208 AGAAAAUGCAGAUG UCU GGGAA 261-279_s 261 162CCCAUCUGCAUUUUCU 209 AGCAGAAAAUGCAG GCU AUGGG 275-293_s 275 163CUGCUGCGGCUGCUGU 210 AUGACAGCAGCCGC CAU AGCAG 276-294_s_C19A 276 164UGCUGCGGCUGCUGUC 211 UAUGACAGCAGCCG AUA CAGCA 276-294_s_C19U 276 165UGCUGCGGCUGCUGUC 212 AAUGACAGCAGCCG AUU CAGCA 278-296_s 278 166CUGCGGCUGCUGUCAU 213 UCGAUGACAGCAGC CGA CGCAG 279-297_s 279 167UGCGGCUGCUGUCAUC 214 AUCGAUGACAGCAG GAU CCGCA 280-298_s_C19A 280 168GCGGCUGCUGUCAUCG 215 UAUCGAUGACAGCA AUA GCCGC 280-298_s_C19U 280 169GCGGCUGCUGUCAUCG 216 AAUCGAUGACAGCA AUU GCCGC 281-299_s 281 170CGGCUGCUGUCAUCGA 217 UGAUCGAUGACAGC UCA AGCCG AD- 282 171GGCUGCUGUCAUCGAU 218 UUGAUCGAUGACAG 11443.1_282-300_s CAA CAGCC AD- 283172 GCUGCUGUCAUCGAUC 219 UUUGAUCGAUGACA 11432.1_283-301_s AAA GCAGC284-302_s_G19A 284 173 CUGCUGUCAUCGAUCA 220 UUUUGAUCGAUGAC AAA AGCAG284-302_s_G19U 284 174 CUGCUGUCAUCGAUCA 221 AUUUGAUCGAUGAC AAU AGCAG AD-285 175 UGCUGUCAUCGAUCAA 222 ACUUUGAUCGAUGA 11441.1_285-303_s AGU CAGCA286-304_s_G19A 286 176 GCUGUCAUCGAUCAAA 223 UACUUUGAUCGAUG GUA ACAGC286-304_s_G19U 286 177 GCUGUCAUCGAUCAAA 224 AACUUUGAUCGAUG GUU ACAGC AD-287 178 CUGUCAUCGAUCAAAG 225 ACACUUUGAUCGAU 11447.1_287-305_s UGU GACAG288-306_s_G19A 288 179 UGUCAUCGAUCAAAGU 226 UACACUUUGAUCGA GUA UGACA288-306_s_G19U 288 180 UGUCAUCGAUCAAAGU 227 AACACUUUGAUCGA GUU UGACA290-308_s_G19A 290 181 UCAUCGAUCAAAGUGU 228 UCCACACUUUGAUC GGA GAUGA290-308_s_G19U 290 182 UCAUCGAUCAAAGUGU 229 ACCACACUUUGAUC GGU GAUGA295-313_s_G19A 295 183 GAUCAAAGUGUGGGAU 230 UACAUCCCACACUU GUA UGAUC295-313_s_G19U 295 184 GAUCAAAGUGUGGGAU 231 AACAUCCCACACUU GUU UGAUC299-317_s_C19U 299 185 AAAGUGUGGGAUGUGC 232 ACAGCACAUCCCAC UGU ACUUUNote that an overhang (e.g. TT, dTsdT) can be added to the 3′ end of anyduplex.

TABLE 4 HAMP modified sequences Table 4 Duplex Start Sense SEQ IDAntisense SEQ ID Target ID Position Name Sense Sequence NO NameAntisense Sequence NO HAMP AD- 2 A-94166.1 AcuGucAcucGGucccAGAdT 233A-94167.1 UCUGGGACCGAGUGA 458 45073 sdT cAGUdTsdT HAMP AD- 7 A-94168.1cAcucGGucccAGAcAccAdTs 234 A-94169.1 UGGUGUCUGGGACCG 459 45079 dTAGUGdTsdT HAMP AD- 16 A-94170.1 ccAGAcAccAGAGcAAGcudT 235 A-94171.1AGCUUGCUCUGGUGU 460 45085 sdT CUGGdTsdT HAMP AD- 43 A-66808.1AGcAGuGGGAcAGccAGAcd 236 A-66809.1 GUCUGGCUGUCCcAC 461 29928 TsdTUGCUdTsdT HAMP AD- 43 A-95618.1 AGcAGuGGGAcAGccAGAAd 237 A-95619.1UUCUGGCUGUCCcAC 462 45674 TsdT UGCUdTsdT HAMP AD- 43 A-95620.1AGcAGuGGGAcAGccAGAud 238 A-95621.1 AUCUGGCUGUCCcAC 463 45680 TsdTUGCUdTsdT HAMP AD- 48 A-95622.1 uGGGAcAGccAGAcAGAcGd 239 A-95623.1CGUCUGUCUGGCUGU 464 45686 TsdT CCcAdTsdT HAMP AD- 48 A-95626.1uGGGAcAGccAGAcAGAcud 240 A-95627.1 AGUCUGUCUGGCUGU 465 45698 TsdTCCcAdTsdT HAMP AD- 48 A-95624.1 uGGGAcAGccAGAcAGAcAd 241 A-95625.1UGUCUGUCUGGCUGU 466 45692 TsdT CCcAdTsdT HAMP AD- 51 A-94701.1GAcAGccAGAcAGAcGGcAd 242 A-94702.1 UGCCGUCUGUCUGGC 467 45354 TsdTUGUCdTsdT HAMP AD- 54 A-66810.1 AGccAGAcAGAcGGcAcGAd 243 A-66811.1UCGUGCCGUCUGUCU 468 29929 TsdT GGCUdTsdT HAMP AD- 55 A-94172.1GccAGAcAGAcGGcAcGAud 244 A-94173.1 AUCGUGCCGUCUGUC 469 45091 TsdTUGGCdTsdT HAMP AD- 59 A-66812.1 GAcAGAcGGcAcGAuGGcAd 245 A-66813.1UGCcAUCGUGCCGUC 470 29930 TsdT UGUCdTsdT HAMP AD- 60 A-66814.1AcAGAcGGcAcGAuGGcAcd 246 A-66815.1 GUGCcAUCGUGCCGU 471 29931 TsdTCUGUdTsdT HAMP AD- 60 A-95628.1 AcAGAcGGcAcGAuGGcAAd 247 A-95629.1UUGCcAUCGUGCCGU 472 45704 TsdT CUGUdTsdT HAMP AD- 60 A-95630.1AcAGAcGGcAcGAuGGcAud 248 A-95631.1 AUGCcAUCGUGCCGU 473 45710 TsdTCUGUdTsdT HAMP AD- 61 A-66816.1 cAGAcGGcAcGAuGGcAcud 249 A-66817.1AGUGCcAUCGUGCCG 474 29932 TsdT UCUGdTsdT HAMP AD- 62 A-98344.1AGACfGGCfACfGAUfGGCfA 250 A-98345.1 AAGUGCCfAUCGUGCC 475 47031CfUfUfdTsdT GUCUdTsdT HAMP AD- 62 A-66818.1 AGAcGGcAcGAuGGcAcuGd 251A-66819.1 cAGUGCcAUCGUGCCG 476 29933 TsdT UCUdTsdT HAMP AD- 62 A-95634.1AGAcGGcAcGAuGGcAcuud 250 A-95635.1 AAGUGCcAUCGUGCC 475 45675 TsdTGUCUdTsdT HAMP AD- 62 A-95632.1 AGAcGGcAcGAuGGcAcuAd 252 A-95633.1uAGUGCcAUCGUGCC 477 45716 TsdT GUCUdTsdT HAMP AD- 63 A-66820.1GAcGGcAcGAuGGcAcuGAd 253 A-66821.1 UcAGUGCcAUCGUGCC 478 29934 TsdTGUCdTsdT HAMP AD- 64 A-66822.1 AcGGcAcGAuGGcAcuGAGd 254 A-66823.1CUcAGUGCcAUCGUGC 479 29935 TsdT CGUdTsdT HAMP AD- 64 A-95638.1AcGGcAcGAuGGcAcuGAud 255 A-95639.1 AUcAGUGCcAUCGUG 480 45687 TsdTCCGUdTsdT HAMP AD- 64 A-95636.1 AcGGcAcGAuGGcAcuGAAd 256 A-95637.1UUcAGUGCcAUCGUG 481 45681 TsdT CCGUdTsdT HAMP AD- 66 A-66824.1GGcAcGAuGGcAcuGAGcud 257 A-66825.1 AGCUcAGUGCcAUCG 482 29936 TsdTUGCCdTsdT HAMP AD- 67 A-98348.1 GCfACfGAUfGGCfACfUfGA 258 A-98349.1AAGCUCfAGUGCCfAU 483 47043 GCfUfUfdTsdT CGUGCdTsdT HAMP AD- 67 A-98346.1GCfACfGAUfGGCfACfUfGA 259 A-98347.1 CfAGCUCfAGUGCCfAU 484 47037GCfUfAdTsdT CGUGCdTsdT HAMP AD- 67 A-66826.1 GcAcGAuGGcAcuGAGcucdT 260A-66827.1 GAGCUcAGUGCcAUC 485 29937 sdT GUGCdTsdT HAMP AD- 67 A-95642.1GcAcGAuGGcAcuGAGcuud 258 A-95643.1 AAGCUcAGUGCcAUCG 483 45699 TsdTUGCdTsdT HAMP AD- 67 A-95640.1 GcAcGAuGGcAcuGAGcuAd 259 A-95641.1uAGCUcAGUGCcAUCG 486 45693 TsdT UGCdTsdT HAMP AD- 68 A-95646.1cAcGAuGGcAcuGAGcucAdT 261 A-95647.1 UGAGCUcAGUGCcAU 487 45711 sdTCGUGdTsdT HAMP AD- 68 A-95648.1 cAcGAuGGcAcuGAGcucudT 262 A-95649.1AGAGCUcAGUGCcAUC 488 45717 sdT GUGdTsdT HAMP AD- 68 A-95644.1cAcGAuGGcAcuGAGcuccdT 263 A-95645.1 GGAGCUcAGUGCcAU 489 45705 sdTCGUGdTsdT HAMP AD- 69 A-95652.1 AcGAuGGcAcuGAGcuccAdT 264 A-95653.1UGGAGCUcAGUGCcA 490 45682 sdT UCGUdTsdT HAMP AD- 69 A-95654.1AcGAuGGcAcuGAGcuccudT 265 A-95655.1 AGGAGCUcAGUGCcA 491 45688 sdTUCGUdTsdT HAMP AD- 69 A-95650.1 AcGAuGGcAcuGAGcucccdT 266 A-95651.1GGGAGCUcAGUGCcA 492 45676 sdT UCGUdTsdT HAMP AD- 70 A-94703.1cGAuGGcAcuGAGcucccAdT 267 A-94704.1 UGGGAGCUcAGUGCc 493 45360 sdTAUCGdTsdT HAMP AD- 71 A-94705.1 GAuGGcAcuGAGcucccAGdT 268 A-94706.1CUGGGAGCUcAGUGC 494 45366 sdT cAUCdTsdT HAMP AD- 72 A-66828.1AuGGcAcuGAGcucccAGAdT 269 A-66829.1 UCUGGGAGCUcAGUG 495 29938 sdTCcAUdTsdT HAMP AD- 73 A-94707.1 uGGcAcuGAGcucccAGAudT 270 A-94708.1AUCUGGGAGCUcAGU 496 45372 sdT GCcAdTsdT HAMP AD- 74 A-98352.1GGCfACfUfGAGCfUfCfCfCfA 271 A-98353.1 AAUCUGGGAGCUCfA 497 47055GAUfUfdTsdT GUGCCdTsdT HAMP AD- 74 A-98350.1 GGCfACfUfGAGCfUfCfCfCfA 272A-98351.1 CfAUCUGGGAGCUCfA 498 47049 GAUfAdTsdT GUGCCdTsdT HAMP AD- 74A-95658.1 GGcAcuGAGcucccAGAuudT 271 A-95659.1 AAUCUGGGAGCUcAG 497 45700sdT UGCCdTsdT HAMP AD- 74 A-66830.1 GGcAcuGAGcucccAGAucdT 273 A-66831.1GAUCUGGGAGCUcAG 499 29939 sdT UGCCdTsdT HAMP AD- 74 A-95656.1GGcAcuGAGcucccAGAuAdT 272 A-95657.1 uAUCUGGGAGCUcAG 500 45694 sdTUGCCdTsdT HAMP AD- 75 A-66832.1 GcAcuGAGcucccAGAucudT 274 A-66833.1AGAUCUGGGAGCUcA 501 29940 sdT GUGCdTsdT HAMP AD- 76 A-98356.1CfACfUfGAGCfUfCfCfCfAGA 275 A-98357.1 AAGAUCUGGGAGCUC 502 47067UfCfUfUfdTsdT fAGUGdTsdT HAMP AD- 76 A-98354.1 CfACfUfGAGCfUfCfCfCfAGA276 A-98355.1 CfAGAUCUGGGAGCUC 503 47061 UfCfUfAdTsdT fAGUGdTsdT HAMPAD- 76 A-95662.1 cAcuGAGcucccAGAucuudTs 275 A-95663.1 AAGAUCUGGGAGCUc502 45712 dT AGUGdTsdT HAMP AD- 76 A-66834.1 cAcuGAGcucccAGAucuGdT 277A-66835.1 cAGAUCUGGGAGCUc 503 29941 sdT AGUGdTsdT HAMP AD- 76 A-95660.1cAcuGAGcucccAGAucuAdTs 276 A-95661.1 uAGAUCUGGGAGCUc 504 45706 dTAGUGdTsdT HAMP AD- 88 A-94174.1 AGAucuGGGccGcuuGccudT 278 A-94175.1AGGcAAGCGGCCcAGA 505 45097 sdT UCUdTsdT HAMP AD- 91 A-94176.1ucuGGGccGcuuGccuccudTs 279 A-94177.1 AGGAGGcAAGCGGCCc 506 45103 dTAGAdTsdT HAMP AD- 116 A-94709.1 ccuccuccucGccAGccuGdTsdT 280 A-94710.1cAGGCUGGCGAGGAG 507 45378 GAGGdTsdT HAMP AD- 117 A-94711.1cuccuccucGccAGccuGAdTs 281 A-94712.1 UcAGGCUGGCGAGGA 508 45383 dTGGAGdTsdT HAMP AD- 118 A-94713.1 uccuccucGccAGccuGAcdTs 282 A-94714.1GUcAGGCUGGCGAGG 509 45388 dT AGGAdTsdT HAMP AD- 120 A-94715.1cuccucGccAGccuGAccAdTs 283 A-94716.1 UGGUcAGGCUGGCGA 510 45393 dTGGAGdTsdT HAMP AD- 121 A-94717.1 uccucGccAGccuGAccAGdTs 284 A-94718.1CUGGUcAGGCUGGCG 511 45355 dT AGGAdTsdT HAMP AD- 122 A-94719.1ccucGccAGccuGAccAGudTs 285 A-94720.1 ACUGGUcAGGCUGGC 512 45361 dTGAGGdTsdT HAMP AD- 123 A-94721.1 cucGccAGccuGAccAGuGdTs 286 A-94722.1cACUGGUcAGGCUGG 513 45367 dT CGAGdTsdT HAMP AD- 126 A-94723.1GccAGccuGAccAGuGGcudT 287 A-94724.1 AGCcACUGGUcAGGC 514 45373 sdTUGGCdTsdT HAMP AD- 132 A-94178.1 cuGAccAGuGGcucuGuuudT 288 A-94179.1AAAcAGAGCcACUGGU 515 45109 sdT cAGdTsdT HAMP AD- 140 A-98360.1UfGGCfUfCfUfGUfUfUfUfCf 289 A-98361.1 UUGUGGGAAAACfAG 516 47032CfCfACfAAdTsdT AGCCfAdTsdT HAMP AD- 140 A-94180.1 uGGcucuGuuuucccAcAAdTs289 A-94181.1 UUGUGGGAAAAcAGA 516 45115 dT GCcAdTsdT HAMP AD- 142A-94182.1 GcucuGuuuucccAcAAcAdTs 290 A-94183.1 UGUUGUGGGAAAAcA 517 45074dT GAGCdTsdT HAMP AD- 146 A-98362.1 UfGUfUfUfUfCfCfCfACfAAC 291A-98363.1 UGUCUGUUGUGGGA 518 47038 fAGACfAdTsdT AAACfAdTsdT HAMP AD- 146A-98364.1 UfGUfUfUfUfCfCfCfACfAAC 292 A-98365.1 AGUCUGUUGUGGGA 519 47044fAGACfUfdTsdT AAACfAdTsdT HAMP AD- 146 A-95666.1 uGuuuucccAcAAcAGAcAdT291 A-95667.1 UGUCUGUUGUGGGA 518 45677 sdT AAAcAdTsdT HAMP AD- 146A-95668.1 uGuuuucccAcAAcAGAcudTs 292 A-95669.1 AGUCUGUUGUGGGA 519 45683dT AAAcAdTsdT HAMP AD- 146 A-95664.1 uGuuuucccAcAAcAGAcGdT 293 A-95665.1CGUCUGUUGUGGGAA 520 45718 sdT AAcAdTsdT HAMP AD- 149 A-94184.1uuucccAcAAcAGAcGGGAdT 294 A-94185.1 UCCCGUCUGUUGUGG 521 45080 sdTGAAAdTsdT HAMP AD- 150 A-94725.1 uucccAcAAcAGAcGGGAcdT 295 A-94726.1GUCCCGUCUGUUGUG 522 45379 sdT GGAAdTsdT HAMP AD- 151 A-66836.1ucccAcAAcAGAcGGGAcAdT 296 A-66837.1 UGUCCCGUCUGUUGU 523 29942 sdTGGGAdTsdT HAMP AD- 152 A-66838.1 cccAcAAcAGAcGGGAcAAdT 297 A-66839.1UUGUCCCGUCUGUUG 524 29943 sdT UGGGdTsdT HAMP AD- 153 A-66840.1ccAcAAcAGAcGGGAcAAcdT 298 A-15142.2 GUUGUCCCGUCUGUU 525 29944 sdTGUGGdTsdT HAMP AD- 153 A-95672.1 ccAcAAcAGAcGGGAcAAudT 299 A-95673.1AUUGUCCCGUCUGUU 526 45695 sdT GUGGdTsdT HAMP AD- 153 A-95670.1ccAcAAcAGAcGGGAcAAAd 300 A-95671.1 UUUGUCCCGUCUGUU 527 45689 TsdTGUGGdTsdT HAMP AD- 154 A-66841.1 cAcAAcAGAcGGGAcAAcudT 301 A-15116.1AGUUGUCCCGUCUGU 528 29945 sdT UGUGdTsdT HAMP AD- 155 A-98366.1ACfAACfAGACfGGGACfAAC 302 A-15182.3 AAGUUGUCCCGUCUG 529 47050 fUfUfdTsdTUUGUdTsdT HAMP AD- 155 A-66842.1 AcAAcAGAcGGGAcAAcuud 302 A-15182.1AAGUUGUCCCGUCUG 529 29946 TsdT UUGUdTsdT HAMP AD- 157 A-98369.1AACfAGACfGGGACfAACfUf 303 A-98370.1 ACfAAGUUGUCCCGUC 530 47062UfGUfdTsdT UGUUdTsdT HAMP AD- 157 A-98367.1 AACfAGACfGGGACfAACfUf 304A-98368.1 UCfAAGUUGUCCCGUC 531 47056 UfGAdTsdT UGUUdTsdT HAMP AD- 157A-95678.1 AAcAGAcGGGAcAAcuuGud 303 A-95679.1 AcAAGUUGUCCCGUC 530 45713TsdT UGUUdTsdT HAMP AD- 157 A-95676.1 AAcAGAcGGGAcAAcuuGAd 304 A-95677.1UcAAGUUGUCCCGUC 531 45707 TsdT UGUUdTsdT HAMP AD- 157 A-95674.1AAcAGAcGGGAcAAcuuGcd 305 A-95675.1 GcAAGUUGUCCCGUC 532 45701 TsdTUGUUdTsdT HAMP AD- 159 A-94727.1 cAGAcGGGAcAAcuuGcAGd 306 A-94728.1CUGcAAGUUGUCCCG 533 45384 TsdT UCUGdTsdT HAMP AD- 160 A-98371.1AGACfGGGACfAACfUfUfGC 307 A-98372.1 UCUGCfAAGUUGUCCC 534 47068 fAGAdTsdTGUCUdTsdT HAMP AD- 160 A-94729.1 AGAcGGGAcAAcuuGcAGAd 307 A-94730.1UCUGcAAGUUGUCCC 534 45389 TsdT GUCUdTsdT HAMP AD- 161 A-98375.1GACfGGGACfAACfUfUfGCf 308 A-98376.1 AUCUGCfAAGUUGUC 535 47033 AGAUfdTsdTCCGUCdTsdT HAMP AD- 161 A-98373.1 GACfGGGACfAACfUfUfGCf 309 A-98374.1UUCUGCfAAGUUGUC 536 47074 AGAAdTsdT CCGUCdTsdT HAMP AD- 161 A-95682.1GAcGGGAcAAcuuGcAGAud 308 A-95683.1 AUCUGcAAGUUGUCC 535 45678 TsdTCGUCdTsdT HAMP AD- 161 A-95680.1 GAcGGGAcAAcuuGcAGAAd 309 A-95681.1UUCUGcAAGUUGUCC 536 45719 TsdT CGUCdTsdT HAMP AD- 161 A-66843.1GAcGGGAcAAcuuGcAGAGd 310 A-66844.1 CUCUGcAAGUUGUCC 537 29947 TsdTCGUCdTsdT HAMP AD- 162 A-98377.1 ACfGGGACfAACfUfUfGCfA 311 A-98378.1UCUCUGCfAAGUUGU 538 47039 GAGAdTsdT CCCGUdTsdT HAMP AD- 162 A-98379.1ACfGGGACfAACfUfUfGCfA 312 A-98380.1 ACUCUGCfAAGUUGU 539 47045 GAGUfdTsdTCCCGUdTsdT HAMP AD- 162 A-95686.1 AcGGGAcAAcuuGcAGAGAd 311 A-95687.1UCUCUGcAAGUUGUC 538 45690 TsdT CCGUdTsdT HAMP AD- 162 A-95688.1AcGGGAcAAcuuGcAGAGud 312 A-95689.1 ACUCUGcAAGUUGUC 539 45696 TsdTCCGUdTsdT HAMP AD- 162 A-95684.1 AcGGGAcAAcuuGcAGAGcd 313 A-95685.1GCUCUGcAAGUUGUC 540 45684 TsdT CCGUdTsdT HAMP AD- 163 A-66845.1cGGGAcAAcuuGcAGAGcud 314 A-66846.1 AGCUCUGcAAGUUGU 541 30016 TsdTCCCGdTsdT HAMP AD- 164 A-94731.1 GGGAcAAcuuGcAGAGcuGd 315 A-94732.1cAGCUCUGcAAGUUG 542 45394 TsdT UCCCdTsdT HAMP AD- 165 A-95690.1GGAcAAcuuGcAGAGcuGcd 316 A-95691.1 GcAGCUCUGcAAGUU 543 45702 TsdTGUCCdTsdT HAMP AD- 165 A-95692.1 GGAcAAcuuGcAGAGcuGAd 317 A-95693.1UcAGCUCUGcAAGUU 544 45708 TsdT GUCCdTsdT HAMP AD- 165 A-95694.1GGAcAAcuuGcAGAGcuGud 318 A-95695.1 AcAGCUCUGcAAGUU 545 45714 TsdTGUCCdTsdT HAMP AD- 166 A-66847.1 GAcAAcuuGcAGAGcuGcAd 319 A-66848.1UGcAGCUCUGcAAGU 546 29949 TsdT UGUCdTsdT HAMP AD- 167 A-94186.1AcAAcuuGcAGAGcuGcAAd 320 A-94187.1 UUGcAGCUCUGcAAG 547 45086 TsdTUUGUdTsdT HAMP AD- 168 A-94733.1 cAAcuuGcAGAGcuGcAAcdT 321 A-94734.1GUUGcAGCUCUGcAA 548 45356 sdT GUUGdTsdT HAMP AD- 169 A-95700.1AAcuuGcAGAGcuGcAAcudT 322 A-95701.1 AGUUGcAGCUCUGcA 549 45685 sdTAGUUdTsdT HAMP AD- 169 A-95698.1 AAcuuGcAGAGcuGcAAcAd 323 A-95699.1UGUUGcAGCUCUGcA 550 45679 TsdT AGUUdTsdT HAMP AD- 169 A-95696.1AAcuuGcAGAGcuGcAAccdT 324 A-95697.1 GGUUGcAGCUCUGcA 551 45720 sdTAGUUdTsdT HAMP AD- 170 A-95706.1 AcuuGcAGAGcuGcAAccudT 325 A-95707.1AGGUUGcAGCUCUGc 552 45703 sdT AAGUdTsdT HAMP AD- 170 A-95704.1AcuuGcAGAGcuGcAAccAdT 326 A-95705.1 UGGUUGcAGCUCUGc 553 45697 sdTAAGUdTsdT HAMP AD- 170 A-95702.1 AcuuGcAGAGcuGcAAcccdT 327 A-95703.1GGGUUGcAGCUCUGc 554 45691 sdT AAGUdTsdT HAMP AD- 189 A-94735.1cAGGAcAGAGcuGGAGccAd 328 A-94736.1 UGGCUCcAGCUCUGU 555 45362 TsdTCCUGdTsdT HAMP AD- 190 A-94737.1 AGGAcAGAGcuGGAGccAG 329 A-94738.1CUGGCUCcAGCUCUG 556 45368 dTsdT UCCUdTsdT HAMP AD- 199 A-94739.1cuGGAGccAGGGccAGcuGd 330 A-94740.1 cAGCUGGCCCUGGCU 557 45374 TsdTCcAGdTsdT HAMP AD- 222 A-94188.1 cccAuGuuccAGAGGcGAAdT 331 A-94189.1UUCGCCUCUGGAAcA 558 45092 sdT UGGGdTsdT HAMP AD- 228 A-95712.1uuccAGAGGcGAAGGAGGu 332 A-95713.1 ACCUCCUUCGCCUCU 559 45721 dTsdTGGAAdTsdT HAMP AD- 228 A-95710.1 uuccAGAGGcGAAGGAGGA 333 A-95711.1UCCUCCUUCGCCUCU 560 45715 dTsdT GGAAdTsdT HAMP AD- 228 A-95708.1uuccAGAGGcGAAGGAGGcd 334 A-95709.1 GCCUCCUUCGCCUCU 561 45709 TsdTGGAAdTsdT HAMP AD- 230 A-94741.1 ccAGAGGcGAAGGAGGcGA 335 A-94742.1UCGCCUCCUUCGCCU 562 45380 dTsdT CUGGdTsdT HAMP AD- 231 A-94743.1cAGAGGcGAAGGAGGcGAG 336 A-94744.1 CUCGCCUCCUUCGCC 563 45385 dTsdTUCUGdTsdT HAMP AD- 232 A-66849.1 AGAGGcGAAGGAGGcGAGA 337 A-66850.1UCUCGCCUCCUUCGC 564 29950 dTsdT CUCUdTsdT HAMP AD- 233 A-94745.1GAGGcGAAGGAGGcGAGAc 338 A-94746.1 GUCUCGCCUCCUUCG 565 45390 dTsdTCCUCdTsdT HAMP AD- 234 A-66851.1 AGGcGAAGGAGGcGAGAcA 339 A-66852.1UGUCUCGCCUCCUUC 566 29951 dTsdT GCCUdTsdT HAMP AD- 235 A-94747.1GGcGAAGGAGGcGAGAcAc 340 A-94748.1 GUGUCUCGCCUCCUU 567 45395 dTsdTCGCCdTsdT HAMP AD- 239 A-95714.1 AAGGAGGcGAGAcAcccAAd 341 A-95715.1UUGGGUGUCUCGCCU 568 45727 TsdT CCUUdTsdT HAMP AD- 239 A-95716.1AAGGAGGcGAGAcAcccAud 342 A-95717.1 AUGGGUGUCUCGCCU 569 45732 TsdTCCUUdTsdT HAMP AD- 239 A-66853.1 AAGGAGGcGAGAcAcccAcd 343 A-66854.1GUGGGUGUCUCGCCU 570 29952 TsdT CCUUdTsdT HAMP AD- 240 A-66855.1AGGAGGcGAGAcAcccAcud 344 A-66856.1 AGUGGGUGUCUCGCC 571 29953 TsdTUCCUdTsdT HAMP AD- 241 A-66857.1 GGAGGcGAGAcAcccAcuud 345 A-66858.1AAGUGGGUGUCUCGC 572 30017 TsdT CUCCdTsdT HAMP AD- 242 A-98383.1GAGGCfGAGACfACfCfCfACf 346 A-95721.2 AAAGUGGGUGUCUCG 573 47057UfUfUfdTsdT CCUCdTsdT HAMP AD- 242 A-98381.1 GAGGCfGAGACfACfCfCfACf 347A-98382.1 CfAAGUGGGUGUCUC 574 47051 UfUfAdTsdT GCCUCdTsdT HAMP AD- 242A-66859.1 GAGGcGAGAcAcccAcuucdT 348 A-66860.1 GAAGUGGGUGUCUCG 575 30018sdT CCUCdTsdT HAMP AD- 242 A-95718.1 GAGGcGAGAcAcccAcuuAdT 347 A-95719.1uAAGUGGGUGUCUCG 576 45737 sdT CCUCdTsdT HAMP AD- 246 A-66861.1cGAGAcAcccAcuuccccAdTs 349 A-66862.1 UGGGGAAGUGGGUG 577 29956 dTUCUCGdTsdT HAMP AD- 247 A-94749.1 GAGAcAcccAcuuccccAudTs 350 A-94750.1AUGGGGAAGUGGGU 578 45357 dT GUCUCdTsdT HAMP AD- 248 A-94751.1AGAcAcccAcuuccccAucdTsdT 351 A-94752.1 GAUGGGGAAGUGGG 579 45363UGUCUdTsdT HAMP AD- 251 A-95722.1 cAcccAcuuccccAucuGcdTsdT 352 A-95723.1GcAGAUGGGGAAGUG 580 45747 GGUGdTsdT HAMP AD- 251 A-95724.1cAcccAcuuccccAucuGAdTsdT 353 A-95725.1 UcAGAUGGGGAAGUG 581 45752GGUGdTsdT HAMP AD- 251 A-95726.1 cAcccAcuuccccAucuGudTsdT 354 A-95727.1AcAGAUGGGGAAGUG 582 45757 GGUGdTsdT HAMP AD- 252 A-66863.1AcccAcuuccccAucuGcAdTsdT 355 A-66864.1 UGcAGAUGGGGAAGU 583 29957GGGUdTsdT HAMP AD- 253 A-98384.1 CfCfCfACfUfUfCfCfCfCfAUfC 356 A-98385.1AUGCfAGAUGGGGAA 584 47063 fUfGCfAUfdTsdT GUGGGdTsdT HAMP AD- 253A-94753.1 cccAcuuccccAucuGcAudTsdT 356 A-94754.1 AUGcAGAUGGGGAAG 58445399 UGGGdTsdT HAMP AD- 255 A-94190.1 cAcuuccccAucuGcAuuudTs 357A-94191.1 AAAUGcAGAUGGGGA 585 45098 dT AGUGdTsdT HAMP AD- 256 A-94755.1AcuuccccAucuGcAuuuudTs 358 A-94756.1 AAAAUGcAGAUGGGG 586 45400 dTAAGUdTsdT HAMP AD- 257 A-94757.1 cuuccccAucuGcAuuuucdTs 359 A-94758.1GAAAAUGcAGAUGGG 587 45381 dT GAAGdTsdT HAMP AD- 258 A-98386.1UfUfCfCfCfCfAUfCfUfGCfAU 360 A-98387.1 AGAAAAUGCfAGAUG 588 47069fUfUfUfCfUfdTsdT GGGAAdTsdT HAMP AD- 258 A-94759.1uuccccAucuGcAuuuucudTs 360 A-94760.1 AGAAAAUGcAGAUGG 588 45401 dTGGAAdTsdT HAMP AD- 261 A-98388.1 CfCfCfAUfCfUfGCfAUfUfUf 361 A-98389.1AGCfAGAAAAUGCfAG 589 47075 UfCfUfGCfUfdTsdT AUGGGdTsdT HAMP AD- 261A-66865.1 cccAucuGcAuuuucuGcudTs 361 A-66866.1 AGcAGAAAAUGcAGA 589 29958dT UGGGdTsdT HAMP AD- 262 A-94761.1 ccAucuGcAuuuucuGcuGdTs 362 A-94762.1cAGcAGAAAAUGcAGA 590 45391 dT UGGdTsdT HAMP AD- 267 A-66867.1uGcAuuuucuGcuGcGGcudT 363 A-66868.1 AGCCGcAGcAGAAAAU 591 29959 sdTGcAdTsdT HAMP AD- 268 A-66869.1 GcAuuuucuGcuGcGGcuGdT 364 A-66870.1cAGCCGcAGcAGAAAA 592 29960 sdT UGCdTsdT HAMP AD- 270 A-66871.1AuuuucuGcuGcGGcuGcudT 365 A-66872.1 AGcAGCCGcAGcAGAA 593 30019 sdTAAUdTsdT HAMP AD- 271 A-94763.1 uuuucuGcuGcGGcuGcuGdT 366 A-94764.1cAGcAGCCGcAGcAGA 594 45396 sdT AAAdTsdT HAMP AD- 272 A-94765.1uuucuGcuGcGGcuGcuGudT 367 A-94766.1 AcAGcAGCCGcAGcAG 595 45358 sdTAAAdTsdT HAMP AD- 273 A-94767.1 uucuGcuGcGGcuGcuGucdT 368 A-94768.1GAcAGcAGCCGcAGcA 596 45364 sdT GAAdTsdT HAMP AD- 274 A-66873.1ucuGcuGcGGcuGcuGucAdT 369 A-66874.1 UGAcAGcAGCCGcAGc 597 29962 sdTAGAdTsdT HAMP AD- 275 A-98390.1 CfUfGCfUfGCfGGCfUfGCfUf 370 A-98391.1AUGACfAGCfAGCCGCf 598 47034 GUfCfAUfdTsdT AGCfAGdTsdT HAMP AD- 275A-94769.1 cuGcuGcGGcuGcuGucAudT 370 A-94770.1 AUGAcAGcAGCCGcAG 598 45370sdT cAGdTsdT HAMP AD- 276 A-98394.1 UfGCfUfGCfGGCfUfGCfUfG 371 A-98395.1AAUGACfAGCfAGCCG 599 47046 UfCfAUfUfdTsdT CfAGCfAdTsdT HAMP AD- 276A-98392.1 UfGCfUfGCfGGCfUfGCfUfG 372 A-98393.1 CfAUGACfAGCfAGCCG 60047040 UfCfAUfAdTsdT CfAGCfAdTsdT HAMP AD- 276 A-95730.1uGcuGcGGcuGcuGucAuudT 371 A-95731.1 AAUGAcAGcAGCCGcA 599 45728 sdTGcAdTsdT HAMP AD- 276 A-95728.1 uGcuGcGGcuGcuGucAuAdT 372 A-95729.1uAUGAcAGcAGCCGcA 601 45722 sdT GcAdTsdT HAMP AD- 276 A-66875.1uGcuGcGGcuGcuGucAucdT 373 A-66876.1 GAUGAcAGcAGCCGcA 602 29963 sdTGcAdTsdT HAMP AD- 278 A-94192.1 cuGcGGcuGcuGucAucGAdT 374 A-94193.1UCGAUGAcAGcAGCCG 603 45104 sdT cAGdTsdT HAMP AD- 279 A-98398.1UfGCfGGCfUfGCfUfGUfCfA 375 A-98399.1 AUCGAUGACfAGCfAG 604 47058UfCfGAUfdTsdT CCGCfAdTsdT HAMP AD- 279 A-66877.1 uGcGGcuGcuGucAucGAudT375 A-66878.1 AUCGAUGAcAGcAGCC 604 29964 sdT GcAdTsdT HAMP AD- 280A-98402.1 GCfGGCfUfGCfUfGUfCfAUf 376 A-98403.1 AAUCGAUGACfAGCfA 60547070 CfGAUfUfdTsdT GCCGCdTsdT HAMP AD- 280 A-98400.1GCfGGCfUfGCfUfGUfCfAUf 377 A-98401.1 CfAUCGAUGACfAGCfA 606 47064CfGAUfAdTsdT GCCGCdTsdT HAMP AD- 280 A-95734.1 GcGGcuGcuGucAucGAuudT 376A-95735.1 AAUCGAUGAcAGcAGC 605 45738 sdT CGCdTsdT HAMP AD- 280 A-95732.1GcGGcuGcuGucAucGAuAd 377 A-95733.1 uAUCGAUGAcAGcAGC 607 45733 TsdTCGCdTsdT HAMP AD- 281 A-98404.1 CfGGCfUfGCfUfGUfCfAUfCf 378 A-98405.1UGAUCGAUGACfAGCf 608 47076 GAUfCfAdTsdT AGCCGdTsdT HAMP AD- 281A-66879.1 cGGcuGcuGucAucGAucAdT 378 A-66880.1 UGAUCGAUGAcAGcA 608 29965sdT GCCGdTsdT HAMP AD- 282 A-98406.1 GGCfUfGCfUfGUfCfAUfCfG 379A-98407.1 UUGAUCGAUGACfAG 609 47035 AUfCfAAdTsdT CfAGCCdTsdT HAMP AD-283 A-98408.1 GCfUfGCfUfGUfCfAUfCfGA 380 A-98409.1 UUUGAUCGAUGACfA 61047041 UfCfAAAdTsdT GCfAGCdTsdT HAMP AD- 283 A-18260.1GcuGcuGucAucGAucAAAdT 380 A-18261.1 UUUGAUCGAUGAcAG 610 30020 sdTcAGCdTsdT HAMP AD- 284 A-98412.1 CfUfGCfUfGUfCfAUfCfGAUf 381 A-98413.1AUUUGAUCGAUGACf 611 47053 CfAAAUfdTsdT AGCfAGdTsdT HAMP AD- 284A-98410.1 CfUfGCfUfGUfCfAUfCfGAUf 382 A-98411.1 UUUUGAUCGAUGACf 61247047 CfAAAAdTsdT AGCfAGdTsdT HAMP AD- 284 A-95738.1cuGcuGucAucGAucAAAudT 381 A-95739.1 AUUUGAUCGAUGAcA 611 45748 sdTGcAGdTsdT HAMP AD- 284 A-95736.1 cuGcuGucAucGAucAAAAdT 382 A-95737.1UUUUGAUCGAUGAcA 612 45743 sdT GcAGdTsdT HAMP AD- 284 A-18284.1cuGcuGucAucGAucAAAGdT 383 A-18285.1 CUUUGAUCGAUGAcA 613 30021 sdTGcAGdTsdT HAMP AD- 285 A-98414.1 UfGCfUfGUfCfAUfCfGAUfCf 384 A-98415.1ACUUUGAUCGAUGAC 614 47059 AAAGUfdTsdT fAGCfAdTsdT HAMP AD- 285 A-18278.3uGcuGucAucGAucAAAGud 384 A-18279.2 ACUUUGAUCGAUGAC 614 11441 TsdTAGcAdTsdT HAMP AD- 286 A-98418.1 GCfUfGUfCfAUfCfGAUfCfA 385 A-98419.1AACUUUGAUCGAUGA 615 47071 AAGUfUfdTsdT CfAGCdTsdT HAMP AD- 286 A-98416.1GCfUfGUfCfAUfCfGAUfCfA 386 A-98417.1 CfACUUUGAUCGAUG 616 47065AAGUfAdTsd TACfAGCdTsdT HAMP AD- 286 A-95742.1 GcuGucAucGAucAAAGuud 385A-95743.1 AACUUUGAUCGAUGA 615 45758 TsdT cAGCdTsdT HAMP AD- 286A-95740.1 GcuGucAucGAucAAAGuAd 386 A-95741.1 uACUUUGAUCGAUGA 617 45753TsdT cAGCdTsdT HAMP AD- 286 A-18288.2 GcuGucAucGAucAAAGuGd 387 A-18289.1cACUUUGAUCGAUGA 616 29968 TsdT cAGCdTsdT HAMP AD- 287 A-98420.1CfUfGUfCfAUfCfGAUfCfAA 388 A-98421.1 ACfACUUUGAUCGAU 618 47077AGUfGUfdTsdT GACfAGdTsdT HAMP AD- 287 A-18290.3 cuGucAucGAucAAAGuGud 388A-18291.1 AcACUUUGAUCGAUG 618 29969 TsdT AcAGdTsdT HAMP AD- 288 A-UGucAucGAucAAAGuGuud 389 A-100243.1 AACACUUUgAuCgAuG 619 48208 100241.2TdT aCadTdT HAMP AD- 288 A-98424.1 UfGUfCfAUfCfGAUfCfAAAG 389 A-98425.1AACfACUUUGAUCGA 619 47042 UfGUfUfdTsdT UGACfAdTsdT HAMP AD- 288 A-uGucAucGAucAAAGuGuud 389 A-100242.1 AACACuuUGauCGAuG 619 48202 100241.1TdT acadTdT HAMP AD- 288 A-98422.1 UfGUfCfAUfCfGAUfCfAAAG 390 A-98423.1CfACfACUUUGAUCGA 620 47036 UfGUfAdTsdT UGACfAdTsdT HAMP AD- 288A-95746.1 uGucAucGAucAAAGuGuud 389 A-95747.1 AAcACUUUGAUCGAU 619 45729TsdT GAcAdTsdT HAMP AD- 288 A-95744.1 uGucAucGAucAAAGuGuAd 390 A-95745.1uAcACUUUGAUCGAU 621 45723 TsdT GAcAdTsdT HAMP AD- 288 A-66881.1uGucAucGAucAAAGuGuGd 391 A-66882.1 cAcACUUUGAUCGAU 620 29970 TsdTGAcAdTsdT HAMP AD- 290 A-98426.1 UfCfAUfCfGAUfCfAAAGUfG 392 A-98427.1UCCfACfACUUUGAUC 622 47048 UfGGAdTsdT GAUGAdTsdT HAMP AD- 290 A-98428.1UfCfAUfCfGAUfCfAAAGUfG 393 A-98429.1 ACCfACfACUUUGAUC 623 47054UfGGUfdTsdT GAUGAdTsdT HAMP AD- 290 A-95752.1 ucAucGAucAAAGuGuGGud 393A-95753.1 ACcAcACUUUGAUCGA 623 45744 TsdT UGAdTsdT HAMP AD- 290A-95750.1 ucAucGAucAAAGuGuGGAd 392 A-95751.1 UCcAcACUUUGAUCGA 622 45739TsdT UGAdTsdT HAMP AD- 290 A-95748.1 ucAucGAucAAAGuGuGGGd 394 A-95749.1CCcAcACUUUGAUCGA 624 45734 TsdT UGAdTsdT HAMP AD- 291 A-98342.1CfAUfCfGAUfCfAAAGUfGUf 395 A-98343.1 UCCCfACfACUUUGAU 625 47005GGGAdTsdT CGAUGdTsdT HAMP AD- 291 A-18268.1 cAucGAucAAAGuGuGGGAd 395A-18269.1 UCCcAcACUUUGAUCG 625 11436 TsdT AUGdTsdT HAMP AD- 291A-18268.1 cAucGAucAAAGuGuGGGAd 395 A-18269.1 UCCcAcACUUUGAUCG 625 11436TsdT AUGdTsdT HAMP AD- 291 A-18268.1 cAucGAucAAAGuGuGGGAd 395 A-18269.1UCCcAcACUUUGAUCG 625 29971 TsdT AUGdTsdT HAMP AD- 292 A-94771.1AucGAucAAAGuGuGGGAud 396 A-94772.1 AUCCcAcACUUUGAUC 626 45376 TsdTGAUdTsdT HAMP AD- 293 A-94773.1 ucGAucAAAGuGuGGGAuG 397 A-94774.1cAUCCcAcACUUUGAU 627 45382 dTsdT CGAdTsdT HAMP AD- 294 A-66883.1cGAucAAAGuGuGGGAuGu 398 A-66884.1 AcAUCCcAcACUUUGA 628 29972 dTsdTUCGdTsdT HAMP AD- 295 A-98432.1 GAUfCfAAAGUfGUfGGGAU 399 A-98433.1AACfAUCCCfACfACUU 629 47066 fGUfUfdTsdT UGAUCdTsdT HAMP AD- 295A-98430.1 GAUfCfAAAGUfGUfGGGAU 400 A-98431.1 CfACfAUCCCfACfACUU 63047060 fGUfAdTsdT UGAUCdTsdT HAMP AD- 295 A-95756.1 GAucAAAGuGuGGGAuGuu399 A-95757.1 AAcAUCCcAcACUUUG 629 45754 dTsdT AUCdTsdT HAMP AD- 295A-95754.1 GAucAAAGuGuGGGAuGuA 400 A-95755.1 uAcAUCCcAcACUUUG 631 45749dTsdT AUCdTsdT HAMP AD- 295 A-66885.1 GAucAAAGuGuGGGAuGuG 401 A-66886.1cAcAUCCcAcACUUUG 630 29973 dTsdT AUCdTsdT HAMP AD- 296 A-95762.1AucAAAGuGuGGGAuGuGu 402 A-95763.1 AcAcAUCCcAcACUUU 632 45730 dTsdTGAUdTsdT HAMP AD- 296 A-95760.1 AucAAAGuGuGGGAuGuGA 403 A-95761.1UcAcAUCCcAcACUUU 633 45724 dTsdT GAUdTsdT HAMP AD- 296 A-95758.1AucAAAGuGuGGGAuGuGc 404 A-95759.1 GcAcAUCCcAcACUUU 634 45759 dTsdTGAUdTsdT HAMP AD- 297 A-94194.1 ucAAAGuGuGGGAuGuGcud 405 A-94195.1AGcAcAUCCcAcACUU 635 45110 TsdT UGAdTsdT HAMP AD- 298 A-94775.1cAAAGuGuGGGAuGuGcuG 406 A-94776.1 cAGcAcAUCCcAcACUU 636 45387 dTsdTUGdTsdT HAMP AD- 299 A-98434.1 AAAGUfGUfGGGAUfGUfGC 407 A-98435.1ACfAGCfACfAUCCCfAC 637 47072 fUfGUfdTsdT fACUUUdTsdT HAMP AD- 299A-95766.1 AAAGuGuGGGAuGuGcuGA 408 A-95767.1 UcAGcAcAUCCcAcACU 638 45740dTsdT UUdTsdT HAMP AD- 299 A-95768.1 AAAGuGuGGGAuGuGcuGu 407 A-95769.1AcAGcAcAUCCcAcACU 637 45745 dTsdT UUdTsdT HAMP AD- 299 A-95764.1AAAGuGuGGGAuGuGcuGc 409 A-95765.1 GcAGcAcAUCCcAcACU 639 45735 dTsdTUUdTsdT HAMP AD- 300 A-66887.1 AAGuGuGGGAuGuGcuGcA 410 A-66888.1UGcAGcAcAUCCcAcAC 640 29974 dTsdT UUdTsdT HAMP AD- 301 A-66889.1AGuGuGGGAuGuGcuGcAA 411 A-66890.1 UUGcAGcAcAUCCcAc 641 29975 dTsdTACUdTsdT HAMP AD- 306 A-94196.1 GGGAuGuGcuGcAAGAcGud 412 A-94197.1ACGUCUUGcAGcAcAU 642 45116 TsdT CCCdTsdT HAMP AD- 307 A-98258.1GGAUfGUfGCfUfGCfAAGAC 413 A-98259.1 CfACGUCUUGCfAGCfA 643 46988fGUfAdTsdT CfAUCCdTsdT HAMP AD- 307 A-94198.1 GGAuGuGcuGcAAGAcGuAd 413A-94199.1 uACGUCUUGcAGcAcA 644 45075 TsdT UCCdTsdT HAMP AD- 309A-98260.1 AUfGUfGCfUfGCfAAGACfG 414 A-98261.1 UCCfACGUCUUGCfAG 645 46994UfAGAdTsdT CfACfAUdTsdT HAMP AD- 309 A-94200.1 AuGuGcuGcAAGAcGuAGAd 414A-94201.1 UCuACGUCUUGcAGcA 646 45081 TsdT cAUdTsdT HAMP AD- 310A-98262.1 UfGUfGCfUfGCfAAGACfGUf 415 A-98263.1 UUCCfACGUCUUGCfA 64747000 AGAAdTsdT GCfACfAdTsdT HAMP AD- 310 A-94202.1 uGuGcuGcAAGAcGuAGAAd415 A-94203.1 UUCuACGUCUUGcAGc 648 45087 TsdT AcAdTsdT HAMP AD- 313A-98264.1 GCfUfGCfAAGACfGUfAGAA 416 A-98265.1 AGGUUCCfACGUCUU 649 47006CfCfUfdTsdT GCfAGCdTsdT HAMP AD- 313 A-94204.1 GcuGcAAGAcGuAGAAccud 416A-94205.1 AGGUUCuACGUCUUG 650 45093 TsdT cAGCdTsdT HAMP AD- 314A-98266.1 CfUfGCfAAGACfGUfAGAACf 417 A-98267.1 CfAGGUUCCfACGUCU 65147011 CfUfAdTsdT UGCfAGdTsdT HAMP AD- 322 A-98268.1CfGUfAGAACfCfUfACfCfUfG 418 A-98269.1 AGGGCfAGGCfAGGUU 652 47016CfCfCfUfdTsdT CCfACGdTsdT HAMP AD- 322 A-94206.1 cGuAGAAccuAccuGcccudTs418 A-94207.1 AGGGcAGGuAGGUUC 653 45099 dT uACGdTsdT HAMP AD- 347A-98270.1 GUfCfCfCfCfUfCfCfCfUfUfCf 419 A-98271.1 AACfAAGGAAGGGAG 65447021 CfUfUfAUfUfdTsdT GGGACdTsdT HAMP AD- 348 A-98272.1UfCfCfCfCfUfCfCfCfUfUfCfC 420 A-98273.1 AAACfAAGGAAGGGAG 655 47026fUfUfAUfUfUfdTsdT GGGAdTsdT HAMP AD- 349 A-98274.1CfCfCfCfUfCfCfCfUfUfCfCfU 421 A-98275.1 CfAAACfAAGGAAGGG 656 46989fUfAUfUfUfAdTsdT AGGGGdTsdT HAMP AD- 350 A-98276.1CfCfCfUfCfCfCfUfUfCfCfUfU 422 A-98277.1 ACfAAACfAAGGAAGG 657 46995fAUfUfUfAUfdTsdT GAGGGdTsdT HAMP AD- 351 A-98278.1CfCfUfCfCfCfUfUfCfCfUfUfA 423 A-98279.1 AACfAAACfAAGGAAG 658 47001UfUfUfAUfUfdTsdT GGAGGdTsdT HAMP AD- 352 A-98282.1CfUfCfCfCfUfUfCfCfUfUfAU 424 A-98283.1 AAACfAAACfAAGGAA 659 47012fUfUfAUfUfUfdTsdT GGGAGdTsdT HAMP AD- 352 A-98280.1CfUfCfCfCfUfUfCfCfUfUfAU 425 A-98281.1 CfAACfAAACfAAGGAA 660 47007fUfUfAUfUfAdTsdT GGGAGdTsdT HAMP AD- 354 A-98284.1CfCfCfUfUfCfCfUfUfAUfUfU 426 A-98285.1 AGGAACfAAACfAAGG 661 47017fAUfUfCfCfUfdTsdT AAGGGdTsdT HAMP AD- 355 A-98286.1CfCfUfUfCfCfUfUfAUfUfUfA 427 A-98287.1 CfAGGAACfAAACfAAG 662 47022UfUfCfCfUfAdTsdT GAAGGdTsdT HAMP AD- 355 A-98288.1CfCfUfUfCfCfUfUfAUfUfUfA 428 A-98289.1 AAGGAACfAAACfAAG 663 47027UfUfCfCfUfUfdTsdT GAAGGdTsdT HAMP AD- 356 A-98292.1CfUfUfCfCfUfUfAUfUfUfAUf 429 A-98293.1 ACfAGGAACfAAACfAA 664 46996UfCfCfUfGUfdTsdT GGAAGdTsdT HAMP AD- 356 A-98290.1CfUfUfCfCfUfUfAUfUfUfAUf 430 A-98291.1 UCfAGGAACfAAACfAA 665 46990UfCfCfUfGAdTsdT GGAAGdTsdT HAMP AD- 357 A-98294.1UfUfCfCfUfUfAUfUfUfAUfU 431 A-98295.1 AGCfAGGAACfAAACfA 666 47002fCfCfUfGCfUfdTsdT AGGAAdTsdT HAMP AD- 358 A-98298.1UfCfCfUfUfAUfUfUfAUfUfCf 432 A-98299.1 AAGCfAGGAACfAAACf 667 47013CfUfGCfUfUfdTsdT AAGGAdTsdT HAMP AD- 358 A-98296.1UfCfCfUfUfAUfUfUfAUfUfCf 433 A-98297.1 CfAGCfAGGAACfAAAC 668 47008CfUfGCfUfAdTsdT fAAGGAdTsdT HAMP AD- 359 A-98302.1CfCfUfUfAUfUfUfAUfUfCfCf 434 A-98303.1 ACfAGCfAGGAACfAAA 669 47023UfGCfUfGUfdTsdT CfAAGGdTsdT HAMP AD- 359 A-98300.1CfCfUfUfAUfUfUfAUfUfCfCf 435 A-98301.1 UCfAGCfAGGAACfAAA 670 47018UfGCfUfGAdTsdT CfAAGGdTsdT HAMP AD- 363 A-98304.1AUfUfUfAUfUfCfCfUfGCfUf 436 A-98305.1 UGGGGCfAGCfAGGAA 671 47028GCfCfCfCfAdTsdT CfAAAUdTsdT HAMP AD- 365 A-98306.1UfUfAUfUfCfCfUfGCfUfGCf 437 A-98307.1 UCUGGGGCfAGCfAGG 672 46991CfCfCfAGAdTsdT AACfAAdTsdT HAMP AD- 366 A-98308.1UfAUfUfCfCfUfGCfUfGCfCf 438 A-98309.1 UUCUGGGGCfAGCfAG 673 46997CfCfAGAAdTsdT GAACfAdTsdT HAMP AD- 369 A-98310.1UfCfCfUfGCfUfGCfCfCfCfAG 439 A-98311.1 AUGUUCUGGGGCfAG 674 47003AACfAUfdTsdT CfAGGAdTsdT HAMP AD- 369 A-94208.1 uccuGcuGccccAGAAcAudTs439 A-94209.1 AUGUUCUGGGGcAGc 674 45105 dT AGGAdTsdT HAMP AD- 370A-98312.1 CfCfUfGCfUfGCfCfCfCfAGA 440 A-98313.1 CfAUGUUCUGGGGCfA 67547009 ACfAUfAdTsdT GCfAGGdTsdT HAMP AD- 370 A-94210.1ccuGcuGccccAGAAcAuAdTs 440 A-94211.1 uAUGUUCUGGGGcAG 676 45111 dTcAGGdTsdT HAMP AD- 373 A-98314.1 GCfUfGCfCfCfCfAGAACfAUf 441 A-98315.1ACCCfAUGUUCUGGG 677 47014 AGGUfdTsdT GCfAGCdTsdT HAMP AD- 373 A-94212.1GcuGccccAGAAcAuAGGudT 441 A-94213.1 ACCuAUGUUCUGGGG 678 45117 sdTcAGCdTsdT HAMP AD- 375 A-98316.1 UfGCfCfCfCfAGAACfAUfAG 442 A-98317.1AGACCCfAUGUUCUG 679 47019 GUfCfUfdTsdT GGGCfAdTsdT HAMP AD- 375A-94214.1 uGccccAGAAcAuAGGucudT 442 A-94215.1 AGACCuAUGUUCUGG 680 45076sdT GGcAdTsdT HAMP AD- 376 A- GccccAGAAcAuAGGucuudT 443 A-100245.1AAGACCuAUGUUCUG 681 48214 100244.1 dT GGGCdTdT HAMP AD- 376 A-GcCCCAGAAcAuAGGucuud 443 A-100247.1 AAGACCuaUGuuCUGG 681 48219 100246.1TdT GGcdTdT HAMP AD- 376 A-98318.1 GCfCfCfCfAGAACfAUfAGGU 443 A-98319.1AAGACCCfAUGUUCUG 682 47024 fCfUfUfdTsdT GGGCdTsdT HAMP AD- 376 A-94216.1GccccAGAAcAuAGGucuudT 443 A-94217.1 AAGACCuAUGUUCUG 681 45082 sdTGGGCdTsdT HAMP AD- 379 A- ccAGAAcAuAGGucuuGGAd 444 A-100249.1UCcAAGACCuAUGUUC 683 48224 100248.1 TdT UCGdTdT HAMP AD- 379 A-ccAGAAcAuAGGucuuGGAd 444 A-100250.1 uCCAAGACCuAUGuUC 684 48187 100248.2TdT uggdTdT HAMP AD- 379 A-98320.1 CfCfAGAACfAUfAGGUfCfUf 444 A-98321.1UCCfAAGACCCfAUGU 685 47029 UfGGAdTsdT UCUGGdTsdT HAMP AD- 379 A-ccAGAAcAuAGGucuuGGAd 444 A-100251.1 uCCAAGACCUaUgUuC 684 48192 100248.3TdT uGgdTdT HAMP AD- 379 A-94218.1 ccAGAAcAuAGGucuuGGAd 444 A-94219.1UCcAAGACCuAUGUUC 684 45088 TsdT UGGdTsdT HAMP AD- 380 A-98322.1CfAGAACfAUfAGGUfCfUfUf 445 A-98323.1 UUCCfAAGACCCfAUG 686 46992GGAAdTsdT UUCUGdTsdT HAMP AD- 380 A-94220.1 cAGAAcAuAGGucuuGGAAd 445A-94221.1 UUCcAAGACCuAUGU 687 45094 TsdT UCUGdTsdT HAMP AD- 381A-98324.1 AGAACfAUfAGGUfCfUfUfG 446 A-98325.1 AUUCCfAAGACCCfAU 688 46998GAAUfdTsdT GUUCUdTsdT HAMP AD- 381 A-94222.1 AGAAcAuAGGucuuGGAAud 446A-94223.1 AUUCcAAGACCuAUG 689 45100 TsdT UUCUdTsdT HAMP AD- 382 A-GAAcAuAGGUCUUGGAAUA 30 A-98136.7 UAUUCCAAGACCUAU 44 48137 100195.1 dTdTGUUCdTdT HAMP AD- 382 A- GAAcAuAGGucuuGGAAuAd 30 A-100228.1UAuUCCAAGaCCuAuG 44 48196 100179.22 TdT uucdTdT HAMP AD- 382 A-98135.8GAACAUAGGUCUUGGAAU 30 A-100218.1 UAUUcCaAgAcCuAuG 44 48195 AdTdT uUcdTdTHAMP AD- 382 A- GAAcAuAGGucuuGGAAuAd 30 A-100229.1 UAUUCCAAGaCCuAuG 4448201 100179.23 TdT uucdTdT HAMP AD- 382 A- GAAcAuAGGucuuGGAAuAd 30A-100230.1 UAUUCCAAgAcCuAuG 44 48207 100179.24 TdT uucdTdT HAMP AD- 382A- GAAcAuAGGucuuGGAAuAd 30 A-100184.1 UAUUCCAAGACCUAu 44 48159 100179.5TdT GuUCdTdT HAMP AD- 382 A- GAAcAuAGGucuuGGAAuAd 30 A-100182.1UAUUCcAAGACCuAuG 44 48147 100179.3 TdT uUCdTdT HAMP AD- 382 A-98135.4GAACAUAGGUCUUGGAAU 30 A-100188.2 UAUUCCAAGACCUAu 44 48161 AdTdT GuUcdTdTHAMP AD- 382 A- GAAcAuAGGucUUGGAAUA 30 A-98136.5 UAUUCCAAGACCUAU 4448172 100193.1 dTdT GUUCdTdT HAMP AD- 382 A- GAAcAuAGGuCUUGGAAUA 30A-100188.3 UAUUCCAAGACCUAu 44 48156 100194.4 dTdT GuUcdTdT HAMP AD- 382A-98135.8 GAACAUAGGUCUUGGAAU 30 A-100218.1 UAUUcCaAgAcCuAuG 44 48195AdTdT uUcdTdT HAMP AD- 382 A- GAAcAuAGGucuuGGAAuAd 30 A-100187.1UAUUCCAAGACCUAU 44 48136 100179.9 TdT GuUcdTdT HAMP AD- 382 A-GAAcAuAGGucuUGGAAUA 30 A-98136.4 UAUUCCAAGACCUAU 44 48166 100192.1 dTdTGUUCdTdT HAMP AD- 382 A- GAAcAuAGGucuuGGAAuAd 30 A-100231.1UAuUCCAAgAcCuAuG 44 48213 100179.25 TdT uucdTdT HAMP AD- 382 A-98135.6GAACAUAGGUCUUGGAAU 30 A-100190.2 UAUUCCAAGACcuAuG 44 48173 AdTdT uUcdTdTHAMP AD- 382 A- GAAcAuAGGucuuGGAAuAd 30 A-100190.1 UAUUCCAAGACcuAuG 4448154 100179.12 TdT uUcdTdT HAMP AD- 382 A- GAAcAuAGGucuuGGAAuAd 30A-100181.1 UAuUCcAAGACCuAuG 44 48141 100179.2 TdT uUCdTdT HAMP AD- 382A- GAAcAuAGGuCUUGGAAUA 30 A-100215.3 UAUUCCAAGACcuAuG 44 48216 100217.3dTsdT uUcdTsdT HAMP AD- 382 A- GAAcAuAGGuCUUGGAAUA 30 A-100183.2UAUUCCAAGACCuAu 44 48180 100194.8 dTdT GuUCdTdT HAMP AD- 382 A-GAAcAUAGGUCUUGGAAU 30 A-98136.8 UAUUCCAAGACCUAU 44 48143 100196.1 AdTdTGUUCdTdT HAMP AD- 382 A- GAAcAuAGGucuuGGAAuAd 30 A-100188.1UAUUCCAAGACCUAu 44 48142 100179.10 TdT GuUcdTdT HAMP AD- 382 A-GAAcAuAGGucuuGGAAuAd 30 A-15168.3 UAUUCCAAGACCUAU 44 48221 18280.13 TsdTGUUCdTsdT HAMP AD- 382 A- GAAcAuAGGucuuGGAAuAd 30 A-98136.2UAUUCCAAGACCUAU 44 48171 100179.7 TdT GUUCdTdT HAMP AD- 382 A-GAAcAuAGGUCUUGGAAUA 30 A-100183.3 UAUUCCAAGACCuAu 44 48145 100195.4 dTdTGuUCdTdT HAMP AD- 382 A- GAAcAuAGGucuuGGAAUA 30 A-98136.3UAUUCCAAGACCUAU 44 48160 100191.1 dTdT GUUCdTdT HAMP AD- 382 A-GAAcAuAGGuCUUGGAAUA 30 A-100190.5 UAUUCCAAGACcuAuG 44 48144 100194.2dTdT uUcdTdT HAMP AD- 382 A-98135.5 GAACAUAGGUCUUGGAAU 30 A-100189.2UAUUCCAAGACCuAu 44 48167 AdTdT GuUcdTdT HAMP AD- 382 A-GAAcAuAGGucuuGGAAuAd 30 A-100186.1 UAUUCCAAGACCUAU 44 48177 100179.8 TdTGUUcdTdT HAMP AD- 382 A- GAAcAuAGGucuuGGAAuAd 30 A-100183.1UAUUCCAAGACCuAu 44 48153 100179.4 TdT GuUCdTdT HAMP AD- 382 A-GAAcAuAGGuCUUGGAAUA 30 A-98136.6 UAUUCCAAGACCUAU 44 48178 100194.1 dTdTGUUCdTdT HAMP AD- 382 A-98135.3 GAACAUAGGUCUUGGAAU 30 A-100187.2UAUUCCAAGACCUAU 44 48155 AdTdT GuUcdTdT HAMP AD- 382 A-GAAcAuAGGuCUUGGAAUA 30 A-100197.1 UAUUCCAAGACcuAuG 44 48174 100194.7dTdT uUCdTdT HAMP AD- 382 A-15167.2 GAACAUAGGUCUUGGAAU 30 A-100215.2UAUUCCAAGACcuAuG 44 48205 AdTsdT uUcdTsdT HAMP AD- 382 A-GAAcAUAGGUCUUGGAAU 30 A-100190.3 UAUUCCAAGACcuAuG 44 48179 100196.2AdTdT uUcdTdT HAMP AD- 382 A- GAAcAuAGGuCUUGGAAUA 30 A-100186.3UAUUCCAAGACCUAU 44 48168 100194.6 dTdT GUUcdTdT HAMP AD- 382 A-98135.2GAACAUAGGUCUUGGAAU 30 A-100186.2 UAUUCCAAGACCUAU 44 48149 AdTdT GUUcdTdTHAMP AD- 382 A- GAAcAuAGGuCUUGGAAUA 30 A-100214.2 UAUUCCAAGACCuAu 4448211 100217.2 dTsdT GuUCdTsdT HAMP AD- 382 A- GAAcAuAGGuCUUGGAAUA 30A-15168.2 UAUUCCAAGACCUAU 44 48200 100217.1 dTsdT GUUCdTsdT HAMP AD- 382A- GAAcAuAGGucuuGGAAuAd 30 A-100205.1 UAuUCcAAGACCuAuG 44 48188100179.20 TdT uUcdTdT HAMP AD- 382 A- GAAcAuAGGucuuGGAAuAd 30 A-100214.1UAUUCCAAGACCuAu 44 48183 18280.10 TsdT GuUCdTsdT HAMP AD- 382 A-GAAcAuAGGuCUUGGAAUA 30 A-100189.3 UAUUCCAAGACCuAu 44 48150 100194.3 dTdTGuUcdTdT HAMP AD- 382 A- GAAcAuAGGuCUUGGAAUA 30 A-100187.3UAUUCCAAGACCUAU 44 48162 100194.5 dTdT GuUcdTdT HAMP AD- 382 A-GAAcAuAGGUCUUGGAAUA 30 A-100197.2 UAUUCCAAGACcuAuG 44 48139 100195.3dTdT uUCdTdT HAMP AD- 382 A-15167.1 GAACAUAGGUCUUGGAAU 30 A-15168.1UAUUCCAAGACCUAU 44 9942 AdTsdT GUUCdTsdT HAMP AD- 382 A-GAAcAuAGGUCUUGGAAUA 30 A-100190.4 UAUUCCAAGACcuAuG 44 48138 100195.2dTdT uUcdTdT HAMP AD- 382 A-18280.2 GAAcAuAGGucuuGGAAuAd 30 A-18304.1uAuUCcAAGACCuAuG 44 11459 TsdT uUCdTsdT HAMP AD- 382 A-GAAcAuAGGucuuGGAAuAd 30 A-100215.1 UAUUCCAAGACcuAuG 44 48189 18280.11TsdT uUcdTsdT HAMP AD- 382 A- GAAcAuAGGucuuGGAAuAd 30 A-100189.1UAUUCCAAGACCuAu 44 48148 100179.11 TdT GuUcdTdT HAMP AD- 382 A-18280.8GAAcAuAGGucuuGGAAuAd 30 A-100212.1 UAuUCcAAGACCuAuG 44 48215 TsdTuUCdTsdT HAMP AD- 382 A- GAAcAuAGGucuuGGAAuAd 30 A-100232.1uAuUCCAAgAcCuAuG 44 48218 100179.26 TdT uucdTdT HAMP AD- 382 A-GAAcAuAGGucuuGGAAuAd 30 A-100180.1 uAuUCcAAGACCuAuG 44 48135 100179.1TdT uUCdTdT HAMP AD- 382 A-98326.1 GAACfAUfAGGUfCfUfUfGG 30 A-98327.1CfAUUCCfAAGACCCfA 690 47004 AAUfAdTsdT UGUUCdTsdT HAMP AD- 382 A-GAAcAuAGGucuuGGAAuAd 30 A-100216.1 uAUUCCAAGACCuAuG 44 48194 18280.12TsdT uUCdTsdT HAMP AD- 382 A- GAAcAuAGGuCdTUdGGdA 447 A-100240.1dTAdTUdCCdAAdGAC 691 48197 100239.1 AdTAdTdT CuAuGuucdTdT HAMP AD- 382A-18280.2 GAAcAuAGGucuuGGAAuAd 30 A-18304.1 uAuUCcAAGACCuAuG 44 11459TsdT uUCdTsdT HAMP AD- 382 A- GAAcAuAGGucuuGGAAuAd 30 A-100201.1uAUUCcAAGACCuAuG 44 48164 100179.16 TdT uUCdTdT HAMP AD- 382 A-GAAcAuAGGucuuGGAAuAd 30 A-100200.1 uAUUCCAAGACCuAuG 44 48158 100179.15TdT uUCdTdT HAMP AD- 382 A- GAAcAcAGGucuuGGAAuAd 448 A-100209.1uAuUCcAAGACCuGuG 692 48204 100208.1 TdT uUCdTdT HAMP AD- 382 A-GAAcAuAGGucuUGGAAUA 30 A-100180.7 uAuUCcAAGACCuAuG 44 48181 100192.2dTdT uUCdTdT HAMP AD- 382 A- GAAcAuAGGucuuGGAAuAuu 449 A-100234.1uAuUCcAAGACCuAuG 706 48223 100233.1 uUCuu HAMP AD- 382 A-GAAcAuAGGucuuGGAAuAd 30 A-100227.1 uAuUCCAAGaCCuAuG 44 48190 100179.21TdT uucdTdT HAMP AD- 382 A- GAAcAuAGGUCUUGGAAUA 30 A-100180.4uAuUCcAAGACCuAuG 44 48163 100195.5 dTdT uUCdTdT HAMP AD- 382 A-GAAcAuAGGucuuGGAAUA 30 A-100180.8 uAuUCcAAGACCuAuG 44 48140 100191.2dTdT uUCdTdT HAMP AD- 382 A- GAAcAuAGGuCUUGGAAUA 30 A-100180.5uAuUCcAAGACCuAuG 44 48169 100194.9 dTdT uUCdTdT HAMP AD- 382 A-18280.9GAAcAuAGGucuuGGAAuAd 30 A-100213.1 uAuUCcAAGACCuAuG 44 48220 TsdTuUcdTsdT HAMP AD- 382 A-15167.3 GAACAUAGGUCUUGGAAU 30 A-18304.6uAuUCcAAGACCuAuG 44 48184 AdTsdT uUCdTsdT HAMP AD- 382 A-GAAcAuAGGucuuGGAAuAd 30 A-100203.1 uAUUCCAAGACCuAuG 44 48176 100179.18TdT uUcdTdT HAMP AD- 382 A- GAAcAuAGGucUUGGAAUA 30 A-100180.6uAuUCcAAGACCuAuG 44 48175 100193.2 dTdT uUCdTdT HAMP AD- 382 A-GAAcAuAGGucuuGGAAuAd 30 A-100198.1 uAUUCCAAGACCUAU 44 48146 100179.13TdT GUUcdTdT HAMP AD- 382 A- GAAcAuAGGucuuGGAAuAd 30 A-100204.1uAuUCcAAGACCuAuG 44 48182 100179.19 TdT uUcdTdT HAMP AD- 382 A-GAAcAuAGGUCUUGGAAuA 30 A- uAuUCcAAGACCuAuG 44 48199 100207.1 dTdT100180.10 uUCdTdT HAMP AD- 382 A- GAAcAUAGGUCUUGGAAU 30 A-100180.3uAuUCcAAGACCuAuG 44 48157 100196.3 AdTdT uUCdTdT HAMP AD- 382 A-GAAcAuAGGucuuGGAAuAd 448 A-100220.1 uAuUCcAAGACCuGuG 692 48206 100219.1TsdT uUCdTsdT HAMP AD- 382 A- GAAcAuAGGuCuuGGAAuA 30 A-100180.9uAuUCcAAGACCuAuG 44 48193 100206.1 dTdT uUCdTdT HAMP AD- 382 A-GAAcAuAGGucuuGGAAuAd 30 A-100199.1 uAUUCCAAGACCUAU 44 48152 100179.14TdT GUUCdTdT HAMP AD- 382 A-98135.7 GAACAUAGGUCUUGGAAU 30 A-100180.2uAuUCcAAGACCuAuG 44 48151 AdTdT uUCdTdT HAMP AD- 382 A-GAAcAuAGGucuuGGAAuAd 30 A-100202.1 uAuUCUAAGACCuAuG 693 48170 100179.17TdT uUCdTdT HAMP AD- 383 A-98328.1 AACfAUfAGGUfCfUfUfGGA 450 A-98329.1UCfAUUCCfAAGACCCf 694 47010 AUfAAdTsdT AUGUUdTsdT HAMP AD- 383 A-94224.1AAcAuAGGucuuGGAAuAAd 450 A-94225.1 UuAUUCcAAGACCuAU 695 45106 TsdTGUUdTsdT HAMP AD- 385 A- CAUAGGUCUUGGAAUAAA 451 A-100225.1UUUUAUUCCAAGACC 696 48222 100224.1 AdTdT UAUGdTdT HAMP AD- 385 A-cAuAGGucuuGGAAuAAAAd 451 A-100223.1 UUUuAUUCcAAGACCu 696 48217 100221.2TdT AUGdTdT HAMP AD- 385 A- cAuAGGucuuGGAAuAAAAd 451 A-100226.1uUUUAuuCCaaGACCU 696 48185 100221.3 TdT augdTdT HAMP AD- 385 A-cAuAGGucuuGGAAuAAAAd 451 A-100222.1 UuUuAuUCcAAGACCu 696 48212 100221.1TdT AuGdTdT HAMP AD- 396 A- GAAuAAAAuGGcuGGuucud 452 A-100253.1AGAACcAGCcAUUUuA 697 48198 100252.1 TdT UUCdTdT HAMP AD- 396 A-GAAuAAAAuGGcuGGuucud 452 A-100255.1 AGAACCAGcCaUuUuA 697 48209 100252.3TdT uUcdTdT HAMP AD- 396 A- GAAuAAAAuGGcuGGuucud 452 A-100254.1AGAACcAGCCAuuUUA 697 48203 100252.2 TdT uucdTdT HAMP AD- 396 A-98330.1GAAUfAAAAUfGGCfUfGGUf 452 A-98331.1 AGAACCfAGCCfAUUU 698 47015UfCfUfdTsdT CfAUUCdTsdT HAMP AD- 396 A-94226.1 GAAuAAAAuGGcuGGuucud 452A-94227.1 AGAACcAGCcAUUUuA 697 45112 TsdT UUCdTsdT HAMP AD- 398A-98332.1 AUfAAAAUfGGCfUfGGUfUf 453 A-98333.1 AAAGAACCfAGCCfAU 699 47020CfUfUfUfdTsdT UUCfAUdTsdT HAMP AD- 398 A-94228.1 AuAAAAuGGcuGGuucuuud453 A-94229.1 AAAGAACcAGCcAUUU 700 45118 TsdT uAUdTsdT HAMP AD- 399A-98334.1 UfAAAAUfGGCfUfGGUfUfCf 454 A-98335.1 AAAAGAACCfAGCCfA 70147025 UfUfUfUfdTsdT UUUCfAdTsdT HAMP AD- 399 A-94230.1uAAAAuGGcuGGuucuuuud 454 A-94231.1 AAAAGAACcAGCcAUU 702 45077 TsdTUuAdTsdT HAMP AD- 402 A-98336.1 AAUfGGCfUfGGUfUfCfUfUf 455 A-98337.1AACfAAAAGAACCfAGC 703 47030 UfUfGUfUfdTsdT CfAUUdTsdT HAMP AD- 402A-94232.1 AAuGGcuGGuucuuuuGuud 455 A-94233.1 AAcAAAAGAACcAGCc 703 45083TsdT AUUdTsdT HAMP AD- 403 A-98338.1 AUfGGCfUfGGUfUfCfUfUfU 456A-98339.1 AAACfAAAAGAACCfA 704 46993 fUfGUfUfUfdTsdT GCCfAUdTsdT HAMPAD- 403 A-94234.1 AuGGcuGGuucuuuuGuuud 456 A-94235.1 AAAcAAAAGAACcAGC704 45089 TsdT cAUdTsdT HAMP AD- 407 A-98340.1 CfUfGGUfUfCfUfUfUfUfGUf457 A-98341.1 UGGAAAACfAAAAGAA 705 46999 UfUfUfCfCfAdTsdT CCfAGdTsdT Itshould be noted that unmodified versions of each of the modifiedsequences shown are included within the scope of the invention

TABLE 5 HAMP unmodified sequences Table 5 Duplex Start AntisenseAntisense SEQ ID Sense SEQ ID Target ID Position Name Sequence NO NameSense Sequence NO HAMP AD- 62 A-98153.1 AGACGGCACGAU 250 A-98154.1AAGUGCCAUCG 475 47121 GGCACUUdTdT UGCCGUCUdTdT HAMP AD- 67 A-98157.1GCACGAUGGCAC 258 A-98158.1 AAGCUCAGUGC 483 47133 UGAGCUUdTdTCAUCGUGCdTdT HAMP AD- 67 A-98155.1 GCACGAUGGCAC 259 A-98156.1UAGCUCAGUGC 486 47127 UGAGCUAdTdT CAUCGUGCdTdT HAMP AD- 74 A-98161.1GGCACUGAGCUCC 271 A-98162.1 AAUCUGGGAGC 497 47145 CAGAUUdTdTUCAGUGCCdTdT HAMP AD- 74 A-98159.1 GGCACUGAGCUCC 272 A-98160.1UAUCUGGGAGC 500 47139 CAGAUAdTdT UCAGUGCCdTdT HAMP AD- 76 A-98165.1CACUGAGCUCCCA 275 A-98166.1 AAGAUCUGGGA 502 47157 GAUCUUdTdTGCUCAGUGdTdT HAMP AD- 76 A-98163.1 CACUGAGCUCCCA 276 A-98164.1UAGAUCUGGGA 504 47151 GAUCUAdTdT GCUCAGUGdTdT HAMP AD- 132 A-98167.1CUGACCAGUGGC 288 A-98168.1 AAACAGAGCCA 515 47163 UCUGUUUdTdTCUGGUCAGdTdT HAMP AD- 140 A-98169.1 UGGCUCUGUUUU 289 A-98170.1UUGUGGGAAA 516 47122 CCCACAAdTdT ACAGAGCCAdTdT HAMP AD- 146 A-98171.1UGUUUUCCCACA 291 A-98172.1 UGUCUGUUGU 518 47128 ACAGACAdTdTGGGAAAACAdTdT HAMP AD- 146 A-98173.1 UGUUUUCCCACA 292 A-98174.1AGUCUGUUGU 519 47134 ACAGACUdTdT GGGAAAACAdTdT HAMP AD- 155 A-98175.1ACAACAGACGGGA 302 A-98176.1 AAGUUGUCCCG 529 47140 CAACUUdTdTUCUGUUGUdTdT HAMP AD- 157 A-98179.1 AACAGACGGGACA 303 A-98180.1ACAAGUUGUCC 530 47152 ACUUGUdTdT CGUCUGUUdTdT HAMP AD- 157 A-98177.1AACAGACGGGACA 304 A-98178.1 UCAAGUUGUCC 531 47146 ACUUGAdTdTCGUCUGUUdTdT HAMP AD- 160 A-98181.1 AGACGGGACAAC 307 A-98182.1UCUGCAAGUUG 534 47158 UUGCAGAdTdT UCCCGUCUdTdT HAMP AD- 161 A-98183.1GACGGGACAACU 309 A-98184.1 UUCUGCAAGUU 536 47164 UGCAGAAdTdTGUCCCGUCdTdT HAMP AD- 161 A-98185.1 GACGGGACAACU 308 A-98186.1AUCUGCAAGUU 535 47123 UGCAGAUdTdT GUCCCGUCdTdT HAMP AD- 162 A-98189.1ACGGGACAACUU 312 A-98190.1 ACUCUGCAAGU 539 47135 GCAGAGUdTdTUGUCCCGUdTdT HAMP AD- 162 A-98187.1 ACGGGACAACUU 311 A-98188.1UCUCUGCAAGU 538 47129 GCAGAGAdTdT UGUCCCGUdTdT HAMP AD- 242 A-98191.1GAGGCGAGACACC 347 A-98192.1 UAAGUGGGUG 576 47141 CACUUAdTdT UCUCGCCUCdTdT HAMP AD- 242 A-98193.1 GAGGCGAGACACC 346 A-98194.1 AAAGUGGGUG 57347147 CACUUUdTdT UCUCGCCUCdT dT HAMP AD- 253 A-98195.1 CCCACUUCCCCAU 356A-98196.1 AUGCAGAUGGG 584 47153 CUGCAUdTdT GAAGUGGGdTdT HAMP AD- 258A-98197.1 UUCCCCAUCUGCA 360 A-98198.1 AGAAAAUGCAG 588 47159 UUUUCUdTdTAUGGGGAAdTdT HAMP AD- 261 A-98199.1 CCCAUCUGCAUU 361 A-98200.1AGCAGAAAAUG 589 47165 UUCUGCUdTdT CAGAUGGGdTdT HAMP AD- 275 A-98201.1CUGCUGCGGCUG 370 A-98202.1 AUGACAGCAGC 598 47124 CUGUCAUdTdTCGCAGCAGdTdT HAMP AD- 276 A-98205.1 UGCUGCGGCUGC 371 A-98206.1AAUGACAGCAG 599 47136 UGUCAUUdTdT CCGCAGCAdTdT HAMP AD- 276 A-98203.1UGCUGCGGCUGC 372 A-98204.1 UAUGACAGCAG 601 47130 UGUCAUAdTdTCCGCAGCAdTdT HAMP AD- 278 A-98207.1 CUGCGGCUGCUG 374 A-98208.1UCGAUGACAGC 603 47142 UCAUCGAdTdT AGCCGCAGdTdT HAMP AD- 279 A-98209.1UGCGGCUGCUGU 375 A-98210.1 AUCGAUGACAG 604 47148 CAUCGAUdTdTCAGCCGCAdTdT HAMP AD- 280 A-98213.1 GCGGCUGCUGUC 376 A-98214.1AAUCGAUGACA 605 47160 AUCGAUUdTdT GCAGCCGCdTdT HAMP AD- 280 A-98211.1GCGGCUGCUGUC 377 A-98212.1 UAUCGAUGACA 607 47154 AUCGAUAdTdTGCAGCCGCdTdT HAMP AD- 281 A-98215.1 CGGCUGCUGUCA 378 A-98216.1UGAUCGAUGAC 608 47166 UCGAUCAdTdT AGCAGCCGdTdT HAMP AD- 282 A-98217.1GGCUGCUGUCAU 379 A-98218.1 UUGAUCGAUGA 609 47125 CGAUCAAdTdTCAGCAGCCdTdT HAMP AD- 283 A-98219.1 GCUGCUGUCAUC 380 A-98220.1UUUGAUCGAU 610 47131 GAUCAAAdTdT GACAGCAGCdT dT HAMP AD- 284 A-98221.1CUGCUGUCAUCG 382 A-98222.1 UUUUGAUCGA 612 47137 AUCAAAAdTdT UGACAGCAGdTdT HAMP AD- 284 A-98223.1 CUGCUGUCAUCG 381 A-98224.1 AUUUGAUCGA 61147143 AUCAAAUdTdT UGACAGCAGdT dT HAMP AD- 285 A-98225.1 UGCUGUCAUCGA 384A-98226.1 ACUUUGAUCGA 614 47149 UCAAAGUdTdT UGACAGCAdTdT HAMP AD- 286A-98229.1 GCUGUCAUCGAU 385 A-98230.1 AACUUUGAUCG 615 47161 CAAAGUUdTdTAUGACAGCdTdT HAMP AD- 286 A-98227.1 GCUGUCAUCGAU 386 A-98228.1UACUUUGAUCG 617 47155 CAAAGUAdTdT AUGACAGCdTdT HAMP AD- 287 A-98231.1CUGUCAUCGAUC 388 A-98232.1 ACACUUUGAUC 618 47167 AAAGUGUdTdTGAUGACAGdTdT HAMP AD- 288 A-98235.1 UGUCAUCGAUCA 389 A-98236.1AACACUUUGAU 619 47132 AAGUGUUdTdT CGAUGACAdTdT HAMP AD- 288 A-98233.1UGUCAUCGAUCA 390 A-98234.1 UACACUUUGAU 621 47126 AAGUGUAdTdTCGAUGACAdTdT HAMP AD- 290 A-98237.1 UCAUCGAUCAAA 392 A-98238.1UCCACACUUUG 622 47138 GUGUGGAdTdT AUCGAUGAdTdT HAMP AD- 290 A-98239.1UCAUCGAUCAAA 393 A-98240.1 ACCACACUUUG 623 47144 GUGUGGUdTdTAUCGAUGAdTdT HAMP AD- 291 A-98151.1 CAUCGAUCAAAG 395 A-98152.1UCCCACACUUU 625 47095 UGUGGGAdTdT GAUCGAUGdTdT HAMP AD- 295 A-98243.1GAUCAAAGUGUG 399 A-98244.1 AACAUCCCACAC 629 47156 GGAUGUUdTdTUUUGAUCdTdT HAMP AD- 295 A-98241.1 GAUCAAAGUGUG 400 A-98242.1UACAUCCCACA 631 47150 GGAUGUAdTdT CUUUGAUCdTdT HAMP AD- 299 A-98245.1AAAGUGUGGGAU 407 A-98246.1 ACAGCACAUCC 637 47162 GUGCUGUdTdTCACACUUUdTdT HAMP AD- 307 A-98067.1 GGAUGUGCUGCA 413 A-98068.1UACGUCUUGCA 644 47078 AGACGUAdTdT GCACAUCCdTdT HAMP AD- 309 A-98069.1AUGUGCUGCAAG 414 A-98070.1 UCUACGUCUUG 646 47084 ACGUAGAdTdTCAGCACAUdTdT HAMP AD- 310 A-98071.1 UGUGCUGCAAGA 415 A-98072.1UUCUACGUCUU 648 47090 CGUAGAAdTdT GCAGCACAdTdT HAMP AD- 313 A-98073.1GCUGCAAGACGU 416 A-98074.1 AGGUUCUACGU 650 47096 AGAACCUdTdTCUUGCAGCdTdT HAMP AD- 314 A-98075.1 CUGCAAGACGUA 417 A-98076.1UAGGUUCUACG 707 47101 GAACCUAdTdT UCUUGCAGdTdT HAMP AD- 322 A-98077.1CGUAGAACCUACC 418 A-98078.1 AGGGCAGGUAG 653 47106 UGCCCUdTdTGUUCUACGdTdT HAMP AD- 347 A-98079.1 GUCCCCUCCCUUC 419 A-98080.1AAUAAGGAAGG 1358 47111 CUUAUUdTdT GAGGGGACdTdT HAMP AD- 348 A-98081.1UCCCCUCCCUUCC 420 A-98082.1 AAAUAAGGAAG 1359 47116 UUAUUUdTdTGGAGGGGAdTdT HAMP AD- 349 A-98083.1 CCCCUCCCUUCCU 421 A-98084.1UAAAUAAGGAA 1360 47079 UAUUUAdTdT GGGAGGGGdTdT HAMP AD- 350 A-98085.1CCCUCCCUUCCUU 422 A-98086.1 AUAAAUAAGGA 1361 47085 AUUUAUdTdTAGGGAGGGdTdT HAMP AD- 351 A-98087.1 CCUCCCUUCCUUA 423 A-98088.1AAUAAAUAAGG 1362 47091 UUUAUUdTdT AAGGGAGGdTdT HAMP AD- 352 A-98089.1CUCCCUUCCUUAU 425 A-98090.1 UAAUAAAUAAG 1363 47097 UUAUUAdTdTGAAGGGAGdTdT HAMP AD- 352 A-98091.1 CUCCCUUCCUUAU 424 A-98092.1AAAUAAAUAAG 1364 47102 UUAUUUdTdT GAAGGGAGdTdT HAMP AD- 354 A-98093.1CCCUUCCUUAUU 426 A-98094.1 AGGAAUAAAUA 1365 47107 UAUUCCUdTdTAGGAAGGGdTdT HAMP AD- 355 A-98095.1 CCUUCCUUAUUU 427 A-98096.1UAGGAAUAAAU 1366 47112 AUUCCUAdTdT AAGGAAGGdTdT HAMP AD- 355 A-98097.1CCUUCCUUAUUU 428 A-98098.1 AAGGAAUAAAU 1367 47117 AUUCCUUdTdTAAGGAAGGdTdT HAMP AD- 356 A-98101.1 CUUCCUUAUUUA 429 A-98102.1ACAGGAAUAAA 1368 47086 UUCCUGUdTdT UAAGGAAGdTdT HAMP AD- 356 A-98099.1CUUCCUUAUUUA 430 A-98100.1 UCAGGAAUAAA 1369 47080 UUCCUGAdTdTUAAGGAAGdTdT HAMP AD- 357 A-98103.1 UUCCUUAUUUAU 431 A-98104.1AGCAGGAAUAA 1370 47092 UCCUGCUdTdT AUAAGGAAdTdT HAMP AD- 358 A-98107.1UCCUUAUUUAUU 432 A-98108.1 AAGCAGGAAUA 1371 47103 CCUGCUUdTdTAAUAAGGAdTdT HAMP AD- 358 A-98105.1 UCCUUAUUUAUU 433 A-98106.1UAGCAGGAAUA 1372 47098 CCUGCUAdTdT AAUAAGGAdTdT HAMP AD- 359 A-98111.1CCUUAUUUAUUC 434 A-98112.1 ACAGCAGGAAU 1373 47113 CUGCUGUdTdTAAAUAAGGdTdT HAMP AD- 359 A-98109.1 CCUUAUUUAUUC 435 A-98110.1UCAGCAGGAAU 1374 47108 CUGCUGAdTdT AAAUAAGGdTdT HAMP AD- 363 A-98113.1AUUUAUUCCUGC 436 A-98114.1 UGGGGCAGCAG 1375 47118 UGCCCCAdTdTGAAUAAAUdTdT HAMP AD- 365 A-98115.1 UUAUUCCUGCUG 437 A-98116.1UCUGGGGCAGC 1376 47081 CCCCAGAdTdT AGGAAUAAdTdT HAMP AD- 366 A-98117.1UAUUCCUGCUGC 438 A-98118.1 UUCUGGGGCAG 1377 47087 CCCAGAAdTdTCAGGAAUAdTdT HAMP AD- 369 A-98119.1 UCCUGCUGCCCCA 439 A-98120.1AUGUUCUGGG 674 47093 GAACAUdTdT GCAGCAGGAdT dT HAMP AD- 370 A-98121.1CCUGCUGCCCCAG 440 A-98122.1 UAUGUUCUGG 676 47099 AACAUAdTdT GGCAGCAGGdTdT HAMP AD- 373 A-98123.1 GCUGCCCCAGAAC 441 A-98124.1 ACCUAUGUUCU 67847104 AUAGGUdTdT GGGGCAGCdTdT HAMP AD- 375 A-98125.1 UGCCCCAGAACAU 442A-98126.1 AGACCUAUGUU 680 47109 AGGUCUdTdT CUGGGGCAdTdT HAMP AD- 376A-98127.1 GCCCCAGAACAUA 443 A-98128.1 AAGACCUAUGU 681 47114 GGUCUUdTdTUCUGGGGCdTdT HAMP AD- 379 A-98129.1 CCAGAACAUAGG 444 A-98130.1UCCAAGACCUA 684 47119 UCUUGGAdTdT UGUUCUGGdTdT HAMP AD- 380 A-98131.1CAGAACAUAGGU 445 A-98132.1 UUCCAAGACCU 687 47082 CUUGGAAdTdTAUGUUCUGdTdT HAMP AD- 381 A-98133.1 AGAACAUAGGUC 446 A-98134.1AUUCCAAGACC 689 47088 UUGGAAUdTdT UAUGUUCUdTdT HAMP AD- 382 A-98135.1GAACAUAGGUCU 30 A-98136.1 UAUUCCAAGAC 44 47094 UGGAAUAdTdT CUAUGUUCdTdTHAMP AD- 382 A-98135.1 GAACAUAGGUCU 30 A-98136.1 UAUUCCAAGAC 44 47094UGGAAUAdTdT CUAUGUUCdTdT HAMP AD- 382 A- GAACACAGGUCU 448 A- UAUUCCAAGAC692 48210 100210.1 UGGAAUAdTdT 100211.1 CUGUGUUCdTdT HAMP AD- 383A-98137.1 AACAUAGGUCUU 450 A-98138.1 UUAUUCCAAGA 695 47100 GGAAUAAdTdTCCUAUGUUdTdT HAMP AD- 396 A-98139.1 GAAUAAAAUGGC 452 A-98140.1AGAACCAGCCA 697 47105 UGGUUCUdTdT UUUUAUUCdTdT HAMP AD- 398 A-98141.1AUAAAAUGGCUG 453 A-98142.1 AAAGAACCAGC 700 47110 GUUCUUUdTdTCAUUUUAUdTdT HAMP AD- 399 A-98143.1 UAAAAUGGCUGG 454 A-98144.1AAAAGAACCAG 702 47115 UUCUUUUdTdT CCAUUUUAdTdT HAMP AD- 402 A-98145.1AAUGGCUGGUUC 455 A-98146.1 AACAAAAGAAC 703 47120 UUUUGUUdTdTCAGCCAUUdTdT HAMP AD- 403 A-98147.1 AUGGCUGGUUCU 456 A-98148.1AAACAAAAGAA 704 47083 UUUGUUUdTdT CCAGCCAUdTdT HAMP AD- 407 A-98149.1CUGGUUCUUUUG 457 A-98150.1 UGGAAAACAAA 705 47089 UUUUCCAdTdTAGAACCAGdTdT

TABLE 6 HAMP single dose screen (Modified Duplexes, Dual LuciferaseAssay) Table 6 Start Duplex Posi- 10 nM 0.1 nM 0.01 nM Target ID tionAvg SD Avg SD Avg SD HAMP AD-45073 2 107.73 0.50 92.94 7.41 HAMPAD-45079 7 110.26 7.14 101.78 5.79 HAMP AD-45085 16 90.81 0.48 96.062.19 HAMP AD-29928 43 102.01 15.80 96.01 1.70 HAMP AD-45674 43 94.814.68 108.44 7.69 HAMP AD-45680 43 109.80 2.04 111.06 5.64 HAMP AD-4568648 89.78 15.04 110.29 0.29 HAMP AD-45698 48 103.33 8.83 112.53 1.57 HAMPAD-45692 48 110.03 6.99 115.05 0.14 HAMP AD-45354 51 111.45 7.56 105.644.49 HAMP AD-29929 54 99.33 11.26 104.08 6.92 HAMP AD-45091 55 116.713.20 102.27 0.81 HAMP AD-29930 59 88.47 0.38 102.18 7.79 HAMP AD-2993160 104.54 3.36 104.80 3.55 HAMP AD-45704 60 142.74 0.80 122.02 1.37 HAMPAD-45710 60 135.87 3.55 129.05 1.72 HAMP AD-29932 61 103.48 6.29 108.361.31 HAMP AD-29933 62 110.13 1.03 104.36 6.68 HAMP AD-45675 62 113.151.01 107.56 0.54 HAMP AD-45716 62 111.06 12.39 113.09 8.16 HAMP AD-2993463 101.68 3.60 96.37 6.01 HAMP AD-29935 64 100.63 8.13 93.98 8.75 HAMPAD-45687 64 103.09 8.83 105.61 3.09 HAMP AD-45681 64 117.87 2.59 111.721.69 HAMP AD-29936 66 98.38 12.53 98.56 13.20 HAMP AD-29937 67 93.412.34 97.50 10.28 HAMP AD-45699 67 47.01 9.59 98.55 3.80 HAMP AD-45693 6784.68 3.15 113.79 5.11 HAMP AD-45711 68 113.03 9.72 108.10 3.83 HAMPAD-45717 68 99.40 12.84 110.38 0.04 HAMP AD-45705 68 110.22 3.84 117.909.96 HAMP AD-45682 69 96.60 3.60 103.41 1.06 HAMP AD-45688 69 100.449.14 104.93 5.18 HAMP AD-45676 69 106.83 9.15 106.73 1.89 HAMP AD-4536070 92.88 0.12 93.73 2.85 HAMP AD-45366 71 92.46 2.58 99.04 0.39 HAMPAD-29938 72 62.08 21.83 75.55 6.85 HAMP AD-45372 73 59.85 2.76 96.315.86 HAMP AD-45700 74 12.85 5.11 63.97 14.79 HAMP AD-29939 74 36.4019.57 67.18 9.10 HAMP AD-45694 74 17.85 4.97 90.43 0.13 HAMP AD-29940 7549.83 6.31 76.05 7.08 HAMP AD-45712 76 32.07 2.85 63.27 3.48 HAMPAD-29941 76 81.10 0.03 97.49 9.32 HAMP AD-45706 76 43.48 6.67 97.60 1.61HAMP AD-45097 88 50.62 0.50 71.18 1.94 HAMP AD-45103 91 53.20 9.02 96.527.45 HAMP AD-45378 116 95.96 1.21 103.17 3.99 HAMP AD-45383 117 99.992.44 104.79 5.38 HAMP AD-45388 118 98.52 4.10 105.96 3.21 HAMP AD-45393120 103.62 5.17 102.44 6.38 HAMP AD-45355 121 73.28 0.51 96.56 1.06 HAMPAD-45361 122 98.67 0.23 99.82 4.47 HAMP AD-45367 123 90.48 1.28 102.754.07 HAMP AD-45373 126 106.01 8.36 99.38 4.05 HAMP AD-45109 132 85.865.55 95.06 3.75 HAMP AD-45115 140 100.97 1.25 90.90 9.28 HAMP AD-45074142 95.53 2.12 95.37 2.74 HAMP AD-45677 146 58.20 5.06 80.37 5.46 HAMPAD-45683 146 67.80 1.12 88.08 7.96 HAMP AD-45718 146 76.16 3.48 100.373.35 HAMP AD-45080 149 52.89 1.28 84.16 0.02 HAMP AD-45379 150 48.973.64 103.43 1.19 HAMP AD-29942 151 88.17 4.85 97.49 7.95 HAMP AD-29943152 43.37 9.93 73.15 23.90 HAMP AD-29944 153 80.38 9.90 92.54 7.85 HAMPAD-45695 153 65.57 2.52 92.72 5.87 HAMP AD-45689 153 72.67 0.78 93.002.67 HAMP AD-29945 154 69.81 13.13 76.49 17.11 HAMP AD-29946 155 75.801.18 80.39 14.15 HAMP AD-45713 157 70.69 1.76 94.45 0.39 HAMP AD-45707157 71.62 6.17 94.94 2.22 HAMP AD-45701 157 79.39 1.97 101.46 0.70 HAMPAD-45384 159 89.86 1.67 102.53 2.37 HAMP AD-45389 160 41.14 0.44 90.293.94 HAMP AD-45678 161 55.04 0.96 76.03 3.63 HAMP AD-45719 161 55.025.94 84.45 2.01 HAMP AD-29947 161 81.45 6.55 89.78 7.92 HAMP AD-45690162 105.99 3.29 97.21 2.61 HAMP AD-45696 162 105.48 0.08 99.13 0.28 HAMPAD-45684 162 96.14 6.48 104.09 2.52 HAMP AD-30016 163 57.89 8.28 90.6014.06 HAMP AD-45394 164 87.68 5.01 108.27 2.32 HAMP AD-45702 165 70.602.02 93.12 5.12 HAMP AD-45708 165 74.75 3.73 98.51 2.26 HAMP AD-45714165 73.26 2.24 102.34 12.47 HAMP AD-29949 166 102.90 8.09 91.62 0.16HAMP AD-45086 167 120.81 3.27 106.79 7.19 HAMP AD-45356 168 81.17 4.4093.13 0.76 HAMP AD-45685 169 114.45 9.16 98.53 0.41 HAMP AD-45679 169105.22 9.07 101.56 5.80 HAMP AD-45720 169 121.03 5.25 110.57 1.75 HAMPAD-45703 170 44.33 1.60 79.12 4.45 HAMP AD-45697 170 46.91 2.65 87.121.48 HAMP AD-45691 170 54.15 1.94 92.73 6.86 HAMP AD-45362 189 40.880.51 88.62 2.54 HAMP AD-45368 190 31.23 1.19 95.59 2.85 HAMP AD-45374199 101.82 3.03 101.10 0.65 HAMP AD-45092 222 87.17 5.48 98.58 1.36 HAMPAD-45721 228 46.67 6.31 81.09 9.13 HAMP AD-45715 228 49.86 3.40 88.144.98 HAMP AD-45709 228 77.17 5.09 98.27 3.31 HAMP AD-45380 230 62.833.09 103.76 1.31 HAMP AD-45385 231 98.28 0.86 102.61 0.12 HAMP AD-29950232 55.13 8.89 67.22 10.51 HAMP AD-45390 233 43.22 3.42 94.61 0.86 HAMPAD-29951 234 37.28 9.48 53.43 13.93 HAMP AD-45395 235 60.56 0.93 96.881.63 HAMP AD-45727 239 41.79 5.36 73.07 5.68 HAMP AD-45732 239 40.158.90 73.60 14.88 HAMP AD-29952 239 97.66 18.17 104.87 4.70 HAMP AD-29953240 86.68 10.48 88.35 7.38 HAMP AD-30017 241 33.76 16.25 60.73 30.76HAMP AD-30018 242 41.44 14.83 70.97 23.75 HAMP AD-45737 242 17.97 4.4971.13 9.76 HAMP AD-29956 246 89.56 4.13 97.82 5.97 HAMP AD-45357 24782.69 2.17 93.22 4.53 HAMP AD-45363 248 93.32 5.91 91.24 0.20 HAMPAD-45747 251 70.65 8.85 97.73 2.26 HAMP AD-45752 251 89.51 3.39 98.352.19 HAMP AD-45757 251 82.94 6.75 102.34 3.76 HAMP AD-29957 252 81.9911.58 93.00 9.35 HAMP AD-45399 253 64.38 0.64 97.91 2.54 HAMP AD-45098255 82.48 2.01 76.84 2.07 HAMP AD-45400 256 41.85 0.69 73.87 3.09 HAMPAD-45381 257 33.48 1.75 76.90 0.24 HAMP AD-45401 258 20.19 1.67 47.653.20 HAMP AD-29958 261 56.65 14.92 84.66 28.04 HAMP AD-45391 262 24.940.82 89.45 2.12 HAMP AD-29959 267 31.48 11.63 63.85 30.99 HAMP AD-29960268 79.91 12.47 93.49 6.68 HAMP AD-30019 270 63.27 10.61 74.99 18.30HAMP AD-45396 271 119.24 2.30 111.41 3.10 HAMP AD-45358 272 94.71 8.18101.07 2.21 HAMP AD-45364 273 84.76 0.19 96.53 5.62 HAMP AD-29962 27476.15 9.81 86.71 13.14 HAMP AD-45370 275 72.03 3.07 96.93 2.90 HAMPAD-45728 276 16.69 2.26 33.05 13.53 HAMP AD-45722 276 14.19 2.15 36.388.98 HAMP AD-29963 276 33.66 10.79 68.80 33.38 HAMP AD-45104 278 68.722.74 87.22 0.49 HAMP AD-29964 279 71.02 18.87 76.03 27.29 HAMP AD-45738280 50.02 8.64 70.44 7.26 HAMP AD-45733 280 57.29 5.28 84.47 5.76 HAMPAD-29965 281 55.85 8.35 72.34 23.30 HAMP AD-30020 283 68.86 8.88 66.0223.24 HAMP AD-45748 284 21.85 1.77 35.95 6.98 HAMP AD-45743 284 29.011.73 42.99 5.69 HAMP AD-30021 284 42.30 7.75 66.28 27.47 HAMP AD-11441285 15.04 8.59 34.60 10.87 63.42 16.67 HAMP AD-45758 286 17.08 0.4333.34 3.43 HAMP AD-45753 286 25.19 4.02 80.83 6.73 HAMP AD-29968 28657.05 12.26 85.22 13.56 HAMP AD-29969 287 81.97 16.19 102.53 21.58 HAMPAD-45729 288 9.67 1.06 32.83 13.93 HAMP AD-45723 288 20.87 3.89 66.574.73 HAMP AD-29970 288 65.21 1.72 84.12 5.99 HAMP AD-45744 290 40.341.92 56.56 8.09 HAMP AD-45739 290 29.46 2.77 67.24 9.80 HAMP AD-45734290 53.39 2.32 83.49 1.19 HAMP AD-11436 291 19.18 8.22 42.74 14.18 76.4323.00 HAMP AD-29971 291 29.02 9.90 52.08 14.09 HAMP AD-45376 292 47.540.51 87.50 1.26 HAMP AD-45382 293 37.05 0.44 93.25 4.03 HAMP AD-29972294 32.08 7.08 53.51 17.12 HAMP AD-45754 295 30.78 4.04 62.74 0.50 HAMPAD-45749 295 48.49 11.81 92.97 1.08 HAMP AD-29973 295 101.69 8.46 97.658.24 HAMP AD-45730 296 78.23 2.00 86.13 5.69 HAMP AD-45724 296 82.074.46 86.67 2.23 HAMP AD-45759 296 92.10 6.68 97.71 2.40 HAMP AD-45110297 69.77 2.74 90.01 2.11 HAMP AD-45387 298 98.34 5.75 108.20 1.85 HAMPAD-45740 299 124.12 4.66 101.03 6.78 HAMP AD-45745 299 131.69 10.22103.23 1.76 HAMP AD-45735 299 111.96 3.19 103.86 6.46 HAMP AD-29974 30034.04 6.78 53.98 24.23 HAMP AD-29975 301 53.80 12.05 67.73 22.08 HAMPAD-45116 306 25.93 2.21 55.49 5.82 HAMP AD-45075 307 19.84 2.98 63.831.45 HAMP AD-45081 309 14.66 0.55 38.67 3.25 HAMP AD-45087 310 11.950.17 29.23 1.61 HAMP AD-45093 313 14.07 0.68 45.44 2.30 HAMP AD-45099322 108.37 1.23 107.51 2.82 HAMP AD-45105 369 102.93 6.40 101.31 4.27HAMP AD-45111 370 117.00 3.72 104.04 2.33 HAMP AD-45117 373 99.33 0.61102.95 0.52 HAMP AD-45076 375 62.84 4.39 90.32 1.71 HAMP AD-45082 37675.58 1.59 95.17 0.21 HAMP AD-45088 379 83.27 12.84 101.84 1.83 HAMPAD-45094 380 99.51 2.23 102.51 2.51 HAMP AD-45100 381 112.68 6.11 107.467.75 HAMP AD-45106 383 138.19 1.98 112.49 0.89 HAMP AD-45112 396 128.116.65 106.21 3.64 HAMP AD-45118 398 116.86 12.51 103.78 4.53 HAMPAD-45077 399 98.64 1.22 103.30 2.55 HAMP AD-45083 402 114.82 2.26 104.500.17 HAMP AD-45089 403 107.59 7.70 103.43 1.69 Data are expressed aspercent of mock or AD-1955.

TABLE 7 HAMP single dose screen (Unmodified Duplexes, Human Endogenous)Table 7 Start Duplex Posi- 10 nM 0.1 nM 0.01 nM Target ID tion Avg SDAvg SD Avg SD HAMP AD-47121 62 22.18 1.49 60.31 16.26 HAMP AD-47133 6727.53 1.12 53.58 2.92 HAMP AD-47127 67 20.45 4.63 54.16 7.57 HAMPAD-47145 74 19.51 6.65 54.67 10.88 HAMP AD-47139 74 20.72 0.17 58.122.90 HAMP AD-47157 76 10.07 0.35 28.24 6.49 HAMP AD-47151 76 12.08 1.8333.95 2.14 HAMP AD-47163 132 8.58 0.51 44.65 17.97 HAMP AD-47122 14025.66 0.45 72.63 2.29 HAMP AD-47128 146 30.88 3.04 64.36 2.04 HAMPAD-47134 146 48.07 0.91 72.33 12.48 HAMP AD-47140 155 15.20 1.25 34.690.30 HAMP AD-47152 157 13.21 6.55 28.17 1.17 HAMP AD-47146 157 14.770.68 32.02 1.58 HAMP AD-47158 160 9.73 1.66 32.92 1.87 HAMP AD-47164 1615.71 0.44 32.90 2.89 HAMP AD-47123 161 7.88 3.02 39.31 19.09 HAMPAD-47135 162 26.84 0.87 74.06 15.78 HAMP AD-47129 162 27.18 1.57 83.6213.59 HAMP AD-47141 242 110.80 16.98 127.17 42.39 HAMP AD-47147 242116.01 9.55 132.52 28.61 HAMP AD-47153 253 34.69 7.47 66.88 2.08 HAMPAD-47159 258 33.41 1.23 57.26 7.97 HAMP AD-47165 261 25.12 0.71 85.706.86 HAMP AD-47124 275 36.35 7.66 87.65 11.44 HAMP AD-47136 276 6.060.70 40.72 13.94 HAMP AD-47130 276 8.76 0.58 46.31 13.29 HAMP AD-47142278 24.10 2.89 56.75 18.44 HAMP AD-47148 279 19.36 1.09 57.95 18.21 HAMPAD-47160 280 8.75 0.73 35.24 4.98 HAMP AD-47154 280 15.01 3.91 36.320.45 HAMP AD-47166 281 11.98 0.47 51.40 12.88 HAMP AD-47125 282 14.621.15 54.37 11.47 HAMP AD-47131 283 8.74 0.45 42.66 12.21 HAMP AD-47137284 9.97 0.73 36.35 5.96 HAMP AD-47143 284 9.66 0.84 39.72 8.37 HAMPAD-47149 285 13.85 0.47 47.21 9.13 HAMP AD-47161 286 7.28 0.89 31.758.03 HAMP AD-47155 286 8.27 0.74 36.03 14.37 HAMP AD-47167 287 8.98 0.1441.61 6.60 HAMP AD-47132 288 9.08 0.17 38.01 4.01 HAMP AD-47126 288 8.593.66 40.28 10.49 HAMP AD-47138 290 41.75 1.27 81.70 14.64 HAMP AD-47144290 60.81 13.34 107.58 15.10 HAMP AD-47095 291 34.79 5.48 58.98 7.36HAMP AD-47156 295 39.09 4.26 90.08 4.38 HAMP AD-47150 295 53.01 9.5899.42 9.61 HAMP AD-47162 299 122.90 8.44 123.74 15.71 HAMP AD-47078 30726.81 9.00 59.79 15.73 HAMP AD-47084 309 31.16 5.91 59.33 19.95 HAMPAD-47090 310 15.07 5.19 49.74 11.70 HAMP AD-47096 313 49.34 9.86 68.640.71 HAMP AD-47101 314 13.36 5.68 38.13 10.64 HAMP AD-47106 322 29.911.99 61.30 1.25 HAMP AD-47111 347 21.57 4.45 46.65 10.06 HAMP AD-47116348 32.95 7.34 65.00 10.06 HAMP AD-47079 349 10.10 2.16 24.36 4.46 HAMPAD-47085 350 8.08 5.13 20.39 9.47 HAMP AD-47091 351 20.73 6.86 42.285.15 HAMP AD-47097 352 10.57 2.97 24.58 3.18 HAMP AD-47102 352 15.487.81 25.60 8.37 HAMP AD-47107 354 50.89 12.39 61.80 6.52 HAMP AD-47112355 42.93 6.42 53.00 3.93 HAMP AD-47117 355 33.82 2.18 60.78 7.57 HAMPAD-47086 356 16.50 3.69 34.88 9.79 HAMP AD-47080 356 13.76 3.39 38.957.09 HAMP AD-47092 357 35.01 5.39 48.61 6.81 HAMP AD-47103 358 45.099.10 66.18 7.81 HAMP AD-47098 358 63.20 11.74 70.69 1.23 HAMP AD-47113359 27.42 9.95 49.88 7.22 HAMP AD-47108 359 30.30 9.89 52.33 12.30 HAMPAD-47118 363 7.45 0.35 19.20 1.31 HAMP AD-47081 365 4.25 1.97 22.94 6.70HAMP AD-47087 366 9.49 2.18 37.51 12.04 HAMP AD-47093 369 4.75 1.3823.36 3.30 HAMP AD-47099 370 5.05 0.01 17.71 7.66 HAMP AD-47104 37332.32 8.82 37.72 8.15 HAMP AD-47109 375 25.45 4.46 35.56 1.25 HAMPAD-47114 376 10.65 4.55 17.30 6.01 HAMP AD-47119 379 7.99 0.50 17.446.45 HAMP AD-47082 380 13.13 1.08 27.19 8.88 HAMP AD-47088 381 5.80 2.7512.26 7.13 HAMP AD-47094 382 5.59 2.35 11.95 7.39 HAMP AD-47094 382 8.633.05 14.02 2.30 22.83 0.56 HAMP AD-48210 382 7.43 7.88 17.06 4.17 30.210.63 HAMP AD-47100 383 3.80 2.75 8.41 3.86 HAMP AD-47105 396 6.56 2.2512.11 4.90 HAMP AD-47110 398 10.42 5.14 21.44 0.24 HAMP AD-47115 3994.86 0.27 9.25 1.57 HAMP AD-47120 402 5.78 0.12 15.68 0.67 HAMP AD-47083403 4.36 1.88 14.49 5.26 HAMP AD-47089 407 17.68 1.22 21.61 6.91 Dataare expressed as percent of mock.

TABLE 8 HAMP single dose screen (Modified Duplexes, Human Endogenous)Table 8 Start Duplex Posi- 10 nM 0.1 nM 0.01 nM Target ID tion Avg SDAvg SD Avg SD HAMP AD-47031 62 15.53 6.23 42.68 4.24 HAMP AD-47043 6721.87 1.62 50.05 4.67 HAMP AD-47037 67 23.85 4.92 53.84 0.43 HAMPAD-47055 74 31.38 2.06 59.08 4.48 HAMP AD-47049 74 30.11 3.10 64.35 3.66HAMP AD-47067 76 8.71 1.38 28.60 2.87 HAMP AD-47061 76 11.78 3.07 29.530.18 HAMP AD-47032 140 37.89 8.04 60.90 3.74 HAMP AD-47038 146 33.922.79 53.24 7.14 HAMP AD-47044 146 39.99 7.45 60.74 5.99 HAMP AD-47050155 14.55 1.46 35.51 0.33 HAMP AD-47062 157 13.42 1.10 45.34 3.82 HAMPAD-47056 157 23.31 0.44 46.02 0.80 HAMP AD-47068 160 24.68 0.67 56.124.66 HAMP AD-47033 161 11.56 4.54 36.94 0.19 HAMP AD-47074 161 9.99 1.0744.47 0.95 HAMP AD-47039 162 63.29 2.38 80.39 15.70 HAMP AD-47045 16286.89 5.22 96.60 14.33 HAMP AD-47057 242 66.74 2.06 82.90 10.89 HAMPAD-47051 242 72.68 0.12 86.34 3.07 HAMP AD-47063 253 26.21 0.40 58.947.25 HAMP AD-47069 258 30.01 3.26 41.02 3.03 HAMP AD-47075 261 30.802.74 75.66 1.48 HAMP AD-47034 275 54.15 10.01 75.48 16.26 HAMP AD-47046276 13.55 0.80 30.18 6.37 HAMP AD-47040 276 18.09 3.87 40.15 14.54 HAMPAD-47058 279 36.00 4.98 64.23 1.93 HAMP AD-47070 280 12.74 1.13 34.849.02 HAMP AD-47064 280 17.08 0.13 49.50 0.21 HAMP AD-47076 281 12.071.81 36.35 5.58 HAMP AD-47035 282 31.01 7.32 61.60 1.15 HAMP AD-47041283 16.92 0.64 39.03 10.75 HAMP AD-47053 284 10.31 0.77 23.40 7.24 HAMPAD-47047 284 12.12 0.18 30.96 7.74 HAMP AD-47059 285 20.79 0.79 45.236.52 HAMP AD-47071 286 15.36 1.48 36.67 8.67 HAMP AD-47065 286 19.450.16 53.77 19.91 HAMP AD-47077 287 9.85 0.40 45.43 2.39 HAMP AD-48208288 9.71 4.88 14.16 3.25 40.26 4.14 HAMP AD-47042 288 9.47 2.61 24.0211.39 HAMP AD-48202 288 11.49 3.71 27.05 3.20 69.29 1.70 HAMP AD-47036288 10.22 1.87 38.40 8.79 HAMP AD-47048 290 38.00 2.44 80.14 9.40 HAMPAD-47054 290 46.82 5.24 87.19 6.81 HAMP AD-47005 291 34.54 2.08 63.8711.34 HAMP AD-11436 291 43.37 7.53 74.23 14.15 HAMP AD-47066 295 37.846.67 66.36 3.03 HAMP AD-47060 295 52.68 4.93 83.68 16.47 HAMP AD-47072299 74.58 22.86 117.51 7.68 HAMP AD-46988 307 39.46 7.63 78.38 1.82 HAMPAD-46994 309 91.00 6.12 100.96 9.30 HAMP AD-47000 310 42.88 7.35 65.346.09 HAMP AD-47006 313 27.81 0.36 71.03 9.01 HAMP AD-47011 314 24.504.38 63.98 14.14 HAMP AD-47016 322 65.73 3.26 90.84 9.34 HAMP AD-47021347 80.76 0.51 86.40 9.24 HAMP AD-47026 348 71.64 5.09 81.58 5.61 HAMPAD-46989 349 90.45 10.46 99.05 11.53 HAMP AD-46995 350 20.68 3.39 75.899.25 HAMP AD-47001 351 74.47 1.50 80.49 21.50 HAMP AD-47012 352 71.8213.01 84.03 5.63 HAMP AD-47007 352 82.28 15.46 89.63 9.85 HAMP AD-47017354 66.26 8.83 100.80 21.89 HAMP AD-47022 355 63.73 4.49 87.39 9.12 HAMPAD-47027 355 68.87 6.64 108.08 32.59 HAMP AD-46996 356 37.91 1.83 48.046.32 HAMP AD-46990 356 41.87 4.92 54.43 6.70 HAMP AD-47002 357 16.190.33 42.98 3.19 HAMP AD-47013 358 22.95 0.97 44.27 7.76 HAMP AD-47008358 20.38 2.71 50.46 16.50 HAMP AD-47023 359 77.40 10.19 95.51 9.29 HAMPAD-47018 359 95.24 14.37 97.19 8.72 HAMP AD-47028 363 28.25 2.86 62.934.48 HAMP AD-46991 365 15.53 2.49 29.41 0.30 HAMP AD-46997 366 31.513.85 48.07 6.21 HAMP AD-47003 369 9.85 2.64 34.31 6.01 HAMP AD-47009 3706.69 1.11 24.11 3.57 HAMP AD-47014 373 55.85 3.19 60.89 10.51 HAMPAD-47019 375 28.54 1.87 49.45 14.83 HAMP AD-48214 376 12.63 3.25 16.691.38 29.21 0.40 HAMP AD-48219 376 15.92 0.02 18.92 0.48 33.17 2.16 HAMPAD-47024 376 19.61 1.81 42.20 5.93 HAMP AD-48224 379 22.20 5.60 33.451.62 52.72 1.81 HAMP AD-48187 379 25.57 5.25 46.92 0.04 73.94 0.20 HAMPAD-47029 379 24.31 0.26 47.37 6.54 HAMP AD-48192 379 19.69 0.78 55.324.62 88.04 0.76 HAMP AD-46992 380 23.41 3.32 37.52 4.13 HAMP AD-46998381 26.55 2.19 49.95 0.87 HAMP AD-48137 382 8.66 0.33 11.24 1.02 26.891.08 HAMP AD-48196 382 6.92 3.59 11.81 1.33 22.33 0.98 HAMP AD-48195 3826.10 2.66 12.50 3.26 26.60 1.17 HAMP AD-48201 382 12.95 1.61 13.01 2.0725.66 6.95 HAMP AD-48207 382 7.91 2.77 13.17 0.62 21.22 1.42 HAMPAD-48159 382 15.26 0.13 13.59 1.45 28.89 4.67 HAMP AD-48147 382 14.280.17 13.65 0.38 25.68 3.81 HAMP AD-48161 382 9.61 3.69 13.77 1.67 22.950.59 HAMP AD-48172 382 11.67 0.41 13.98 1.39 27.28 5.22 HAMP AD-48156382 12.14 0.96 14.06 1.75 28.85 2.18 HAMP AD-48195 382 6.81 0.62 14.140.37 27.99 0.61 HAMP AD-48136 382 10.81 4.42 14.16 1.41 29.89 1.13 HAMPAD-48166 382 8.74 2.41 14.22 0.42 25.21 2.09 HAMP AD-48213 382 8.35 1.6114.49 3.71 22.38 0.33 HAMP AD-48173 382 12.84 3.32 14.51 0.96 27.87 5.30HAMP AD-48154 382 9.93 2.07 14.80 0.75 23.47 0.94 HAMP AD-48141 38212.73 2.32 14.92 0.32 26.97 5.76 HAMP AD-48216 382 10.39 0.95 15.18 2.1122.38 1.02 HAMP AD-48180 382 7.71 0.47 15.20 1.30 29.01 1.11 HAMPAD-48143 382 7.00 2.73 15.44 1.88 24.35 2.09 HAMP AD-48142 382 10.103.42 15.50 0.90 25.84 2.08 HAMP AD-48221 382 9.54 1.43 15.56 0.24 23.591.06 HAMP AD-48171 382 13.46 5.06 15.67 0.63 26.44 6.58 HAMP AD-48145382 10.87 0.68 15.70 2.69 28.65 2.93 HAMP AD-48160 382 10.77 0.26 15.741.29 28.12 4.11 HAMP AD-48144 382 9.88 0.46 15.75 0.94 33.60 0.84 HAMPAD-48167 382 10.83 1.48 15.87 3.03 24.90 2.15 HAMP AD-48177 382 15.052.15 15.87 0.86 28.68 8.05 HAMP AD-48153 382 12.07 5.83 15.92 1.04 31.193.85 HAMP AD-48178 382 11.02 0.05 16.06 0.45 27.12 2.27 HAMP AD-48155382 12.92 0.25 16.32 4.70 27.64 0.32 HAMP AD-48174 382 11.50 2.09 16.390.74 27.90 4.46 HAMP AD-48205 382 7.46 7.62 16.39 2.29 24.17 1.56 HAMPAD-48179 382 10.80 3.42 16.50 0.71 27.82 3.57 HAMP AD-48168 382 12.144.14 16.63 1.58 27.25 1.24 HAMP AD-48149 382 10.42 0.41 16.71 3.88 28.301.91 HAMP AD-48211 382 9.46 4.30 16.80 1.44 25.08 0.20 HAMP AD-48200 3829.05 1.30 16.97 2.20 28.99 0.38 HAMP AD-48188 382 11.09 3.14 16.99 2.4132.42 1.58 HAMP AD-48183 382 9.79 11.87 17.20 0.54 42.02 0.63 HAMPAD-48150 382 9.99 8.19 17.30 2.27 35.68 0.32 HAMP AD-48162 382 8.48 2.9617.38 1.26 29.63 0.85 HAMP AD-48139 382 10.35 1.92 17.78 0.51 36.00 2.79HAMP AD-9942 382 8.96 1.21 18.03 1.85 30.09 0.13 HAMP AD-48138 382 10.062.32 18.04 1.88 26.97 1.56 HAMP AD-11459 382 7.07 0.09 18.93 3.13 HAMPAD-48189 382 8.39 0.33 19.16 0.02 26.30 0.33 HAMP AD-48148 382 10.681.22 19.23 3.07 32.33 1.11 HAMP AD-48215 382 12.87 3.87 19.50 0.69 35.593.70 HAMP AD-48218 382 12.77 1.57 22.32 2.42 48.01 5.44 HAMP AD-48135382 13.43 1.45 26.06 4.80 57.92 1.12 HAMP AD-47004 382 10.04 1.61 26.683.90 HAMP AD-48194 382 13.40 1.06 27.15 1.41 44.44 2.80 HAMP AD-48197382 13.40 6.22 27.28 1.85 47.77 5.73 HAMP AD-11459 382 14.32 0.61 28.202.18 50.13 0.65 HAMP AD-48164 382 16.52 2.52 28.92 3.09 55.94 0.41 HAMPAD-48158 382 11.63 2.57 29.78 1.47 51.55 0.47 HAMP AD-48204 382 11.5313.16 29.90 1.81 68.66 2.16 HAMP AD-48181 382 11.31 3.03 30.04 2.0765.39 3.38 HAMP AD-48223 382 12.35 5.46 30.39 3.74 60.92 1.91 HAMPAD-48190 382 10.09 1.16 30.78 0.29 61.44 0.86 HAMP AD-48163 382 13.432.71 31.64 4.03 60.73 1.22 HAMP AD-48140 382 11.77 2.88 31.73 2.21 57.590.57 HAMP AD-48169 382 12.50 0.13 32.00 4.43 60.20 3.22 HAMP AD-48220382 15.52 3.73 32.05 1.48 58.71 2.57 HAMP AD-48184 382 13.23 0.16 33.253.63 56.78 2.43 HAMP AD-48176 382 16.68 3.88 34.04 1.12 68.51 2.16 HAMPAD-48175 382 13.60 9.49 34.34 2.07 63.89 2.17 HAMP AD-48146 382 13.164.10 35.07 0.23 61.14 1.09 HAMP AD-48182 382 13.71 8.96 36.24 0.98 71.730.19 HAMP AD-48199 382 11.16 0.69 36.33 1.26 66.19 0.94 HAMP AD-48157382 11.27 0.12 36.54 2.61 71.05 0.88 HAMP AD-48206 382 10.51 2.71 36.794.47 61.74 0.21 HAMP AD-48193 382 13.00 3.25 37.58 2.12 73.07 0.26 HAMPAD-48152 382 21.49 5.83 39.17 6.15 68.81 10.01 HAMP AD-48151 382 13.6210.83 39.31 6.55 66.68 1.47 HAMP AD-48170 382 14.54 2.27 47.27 4.0170.43 2.22 HAMP AD-47010 383 41.47 1.29 64.00 4.87 HAMP AD-48222 3859.49 0.13 10.82 1.20 18.78 2.86 HAMP AD-48217 385 14.39 3.84 14.26 0.5519.91 0.71 HAMP AD-48185 385 14.22 4.44 18.35 1.29 37.50 0.03 HAMPAD-48212 385 20.56 4.86 22.61 3.43 26.13 0.41 HAMP AD-48198 396 8.3123.06 13.10 3.95 50.72 1.39 HAMP AD-48209 396 8.76 5.65 14.85 3.44 33.812.06 HAMP AD-48203 396 9.38 2.78 15.08 2.34 35.67 2.38 HAMP AD-47015 39616.43 2.26 30.67 1.14 HAMP AD-47020 398 50.18 1.59 68.91 17.54 HAMPAD-47025 399 13.04 0.87 19.74 1.31 HAMP AD-47030 402 5.12 0.55 12.720.67 HAMP AD-46993 403 5.82 2.21 12.55 1.15 HAMP AD-46999 407 11.34 1.3515.21 1.41 Data are expressed as percent of mock.

TABLE 9 HAMP dose-response (Dual Luciferase, HepG2, Cyno primaryhepatocytes; Unmodified & Modified duplexes) Table 9 Start Duplex posi-Modification IC50 (nM) Target ID tion status Luc HepG2 Cyno HAMPAD-29939 74 Modified 0.288 HAMP AD-45700 74 Modified 0.752 HAMP AD-2994075 Modified 0.929 HAMP AD-29943 152 Modified 0.567 HAMP AD-29950 232Modified 1.527 HAMP AD-29951 234 Modified 0.408 HAMP AD-30017 241Modified 0.163 HAMP AD-30018 242 Modified 0.517 HAMP AD-29959 267Modified 0.147 HAMP AD-29963 276 Modified 0.155 HAMP AD-45722 276Modified 0.299 HAMP AD-29965 281 Modified 1.149 HAMP AD-30020 283Modified 39.122 HAMP AD-30021 284 Modified 0.308 HAMP AD-11441 285Modified 0.042 0.135 0.027 HAMP AD-11458 285 Modified 0.358 HAMPAD-45729 288 Modified 0.068 0.016 HAMP AD-48208 288 Modified 0.012 0.016HAMP AD-11436 291 Modified 0.054 >10 nM HAMP AD-11453 291 Modified 0.134HAMP AD-29971 291 Modified 0.108 HAMP AD-29972 294 Modified 0.154 HAMPAD-29974 300 Modified 0.137 HAMP AD-29975 301 Modified 1.392 HAMPAD-45081 309 Modified >10 nM HAMP AD-45087 310 Modified >10 nM HAMPAD-45093 313 Modified >10 nM HAMP AD-29979 352 Modified >10 nM HAMPAD-45750 352 Modified 1.558 HAMP AD-45755 352 Modified 0.296 HAMPAD-45725 355 Modified >10 nM HAMP AD-29981 357 Modified >10 nM HAMPAD-45761 359 Modified >10 nM HAMP AD-45377 364 Modified >10 nM HAMPAD-29982 365 Modified 1.723 HAMP AD-29983 366 Modified >10 nM HAMPAD-47099 370 Unmodified 0.017 0.081 HAMP AD-47114 376 Unmodified 0.0080.036 HAMP AD-48214 376 Modified 0.008 1.575 HAMP AD-47119 379Unmodified 0.004 0.040 HAMP AD-47088 381 Unmodified 0.007 >10 nM HAMPAD-11442 382 Modified 0.028 0.010 HAMP AD-11459 382 Unmodified 0.0380.045 HAMP AD-45062 382 Modified 0.088 0.030 HAMP AD-47094 382Unmodified 0.005 0.039 HAMP AD-48141 382 Modified 0.004 0.023 HAMPAD-48147 382 Modified 0.007 0.008 HAMP AD-48154 382 Modified 0.006 0.019HAMP AD-48189 382 Modified 0.005 0.145 HAMP AD-48195 382 Modified 0.0110.009 HAMP AD-48196 382 Modified 0.017 0.031 HAMP AD-48201 382 Modified0.007 0.009 HAMP AD-48205 382 Modified 0.014 0.022 HAMP AD-48207 382Modified 0.007 0.017 HAMP AD-48213 382 Modified 0.009 0.027 HAMPAD-48216 382 Modified 0.014 0.035 HAMP AD-47100 383 Unmodified 0.0070.172 HAMP AD-48217 385 Modified 0.028 0.021 HAMP AD-47105 396Unmodified 0.005 >10 nM HAMP AD-48209 396 Modified 0.013 >10 nM HAMPAD-47115 399 Unmodified 0.007 >10 nM HAMP AD-47030 402 Modified0.015 >10 nM HAMP AD-47120 402 Unmodified 0.006 >10 nM HAMP AD-46993 403Modified 0.015 >10 nM HAMP AD-47083 403 Unmodified 0.007 >10 nM HAMPAD-46999 407 Modified 0.011 >10 nM

Tables 10A and 10B: Secondary Target sequences

TABLE 10A Duplex Start Sense SEQ Antisense SEQ ID Target ID PositionName Sense Sequence ID NO Name Antisense Sequence NO HFE2 AD- 177 A-AGAGuAGGGAAucAu 31 A- AGCcAUGAUUCCCuAC 33 47391 98855.1 GGcudTsdT98856.1 UCUdTsdT HFE2 AD- 193 A- GcuGGAGAAuuGGAu 708 A- UGCuAUCcAAUUCUCc753 47397 98857.1 AGcAdTsdT 98858.1 AGCdTsdT HFE2 AD- 195 A-uGGAGAAuuGGAuA 709 A- UCUGCuAUCcAAUUCU 754 47403 98859.1 GcAGAdTsdT98860.1 CcAdTsdT HFE2 AD- 199 A- GAAuuGGAuAGcAGA 710 A- UuACUCUGCuAUCcAA755 47409 98861.1 GuAAdTsdT 98862.1 UUCdTsdT HFE2 AD- 200 A-AAuuGGAuAGcAGAG 711 A- AUuACUCUGCuAUCcA 756 47415 98863.1 uAAudTsdT98864.1 AUUdTsdT HFE2 AD- 206 A- AuAGcAGAGuAAuGu 712 A- UcAAAcAUuACUCUGC757 47421 98865.1 uuGAdTsdT 98866.1 uAUdTsdT HFE2 AD- 211 A-AGAGuAAuGuuuGAcc 713 A- AGAGGUcAAAcAUuAC 758 47427 98867.1 ucudTsdT98868.1 UCUdTsdT HFE2 AD- 244 A- ucAuAuuuAAGAAcAu 714 A-UGcAUGUUCUuAAAuA 759 47433 98869.1 GcAdTsdT 98870.1 UGAdTsdT HFE2 AD-257 A- cAuGcAGGAAuGcAuu 715 A- AUcAAUGcAUUCCUGc 760 47392 98871.1GAudTsdT 98872.1 AUGdTsdT HFE2 AD- 261 A- cAGGAAuGcAuuGAuc 716 A-UCUGAUcAAUGcAUUC 761 47398 98873.1 AGAdTsdT 98874.1 CUGdTsdT HFE2 AD-290 A- GGcuGAGGuGGAuAA 717 A- AAGAUuAUCcACCUcA 762 47404 98875.1ucuudTsdT 98876.1 GCCdTsdT HFE2 AD- 360 A- uccAGuuuGucGAuucA 718 A-UUUGAAUCGAcAAAC 763 47410 98877.1 AAdTsdT 98878.1 UGGAdTsdT HFE2 AD- 367A- uGucGAuucAAAcuGcu 719 A- UuAGcAGUUUGAAUCG 764 47416 98879.1 AAdTsdT98880.1 AcAdTsdT HFE2 AD- 404 A- GAuccAAGcuGccuAc 720 A-AAUGuAGGcAGCUUGG 765 47422 98881.1 AuudTsdT 98882.1 AUCdTsdT HFE2 AD-415 A- ccuAcAuuGGcAcAAcu 721 A- AuAGUUGUGCcAAUGu 766 47428 98883.1AudTsdT 98884.1 AGGdTsdT HFE2 AD- 417 A- uAcAuuGGcAcAAcuA 722 A-UuAuAGUUGUGCcAAU 767 47434 98885.1 uAAdTsdT 98886.1 GuAdTsdT HFE2 AD-472 A- ucAAGGuAGcAGAGG 723 A- AcAUCCUCUGCuACCU 768 47393 98887.1AuGudTsdT 98888.1 UGAdTsdT HFE2 AD- 585 A- GGAGcuAuAAccAuuG 724 A-uAUcAAUGGUuAuAGC 769 47399 98889.1 AuAdTsdT 98890.1 UCCdTsdT HFE2 AD-587 A- AGcuAuAAccAuuGAu 725 A- AGuAUcAAUGGUuAuA 770 47405 98891.1AcudTsdT 98892.1 GCUdTsdT HFE2 AD- 638 A- GGAAGAuGcuuAcuuc 726 A-AUGGAAGuAAGcAUCU 771 47417 98895.1 cAudTsdT 98896.1 UCCdTsdT HFE2 AD-642 A- GAuGcuuAcuuccAuucc 727 A- AGGAAUGGAAGuAAGc 772 47423 98897.1udTsdT 98898.1 AUCdTsdT HFE2 AD- 646 A- cuuAcuuccAuuccuGuG 728 A-AcAcAGGAAUGGAAGu 773 47429 98899.1 udTsdT 98900.1 AAGdTsdT HFE2 AD- 656A- uuccuGuGucuuuGAuG 729 A- AAcAUcAAAGAcAcAG 774 47435 98901.1 uudTsdT98902.1 GAAdTsdT HFE2 AD- 657 A- uccuGuGucuuuGAuGu 730 A-AAAcAUcAAAGAcAcA 775 47394 98903.1 uudTsdT 98904.1 GGAdTsdT HFE2 AD- 678A- AuuucuGGuGAucccAA 731 A- AGUUGGGAUcACcAGA 776 47400 98905.1 cudTsdT98906.1 AAUdTsdT HFE2 AD- 1121 A- ccAuuuAcuGcAGAuuu 732 A-UGAAAUCUGcAGuAAA 777 47406 98907.1 cAdTsdT 98908.1 UGGdTsdT HFE2 AD-1151 A- uuAGAGGucAuGAAG 733 A- AAACCUUcAUGACCUC 778 47412 98909.1GuuudTsdT 98910.1 uAAdTsdT HFE2 AD- 1152 A- uAGAGGucAuGAAGG 734 A-AAAACCUUcAUGACCU 779 47418 98911.1 uuuudTsdT 98912.1 CuAdTsdT HFE2 AD-1203 A- uuAAGAGGcAAGAGc 735 A- UUcAGCUCUUGCCUCU 780 47424 98913.1uGAAdTsdT 98914.1 uAAdTsdT HFE2 AD- 1228 A- AGAcAuGAucAuuAGc 736 A-AUGGCuAAUGAUcAUG 781 47430 98915.1 cAudTsdT 98916.1 UCUdTsdT HFE2 AD-1230 A- AcAuGAucAuuAGccA 737 A- UuAUGGCuAAUGAUcA 782 47436 98917.1uAAdTsdT 98918.1 UGUdTsdT HFE2 AD- 1233 A- uGAucAuuAGccAuAA 738 A-UUCUuAUGGCuAAUGA 783 47395 98919.1 GAAdTsdT 98920.1 UcAdTsdT HFE2 AD-1272 A- AuuAGGGAAAGAAG 739 A- AuAGACUUCUUUCCCu 784 47401 98921.1ucuAudTsdT 98922.1 AAUdTsdT HFE2 AD- 1273 A- uuAGGGAAAGAAGuc 740 A-AAuAGACUUCUUUCCC 785 47407 98923.1 uAuudTsdT 98924.1 uAAdTsdT HFE2 AD-1273 A- uuAGGGAAAGAAGu 740 A- AAuAGACUUCUUUCCC 785 51740 107281.4CuAuUdTsdT 107275.3 uAadTsdT HFE2 AD- 1273 A- uuAGGGAAAGAAGuc 740 A-AAuAGACUuCUuUCcCu 785 51747 107280.6 uAuUdTsdT 107277.2 AAdTsdT HFE2 AD-1273 A- uuAGGGAAAGAAGu 740 A- AAuAGACUuCUuUCCC 785 51744 107281.5CuAuUdTsdT 107276.3 uAAdTsdT HFE2 AD- 1273 A- uuAGGGAAAGAAGuc 740 A-AAuAGACUUCUuUCCC 785 51731 107280.2 uAuUdTsdT 107273.2 uAAdTsdT HFE2 AD-1273 A- uuAGGGAAAGAAGu 740 A- AAuAGACUUCUUUCcC 785 51736 107281.3CuAuUdTsdT 107274.3 uAAdTsdT HFE2 AD- 1273 A- uuAGGGAAAGAAGu 740 A-AAuAGACUUCUuUCCC 785 51732 107281.2 CuAuUdTsdT 107273.3 uAAdTsdT HFE2AD- 1273 A- uuAGGGAAAGAAGuc 740 A- AAuAGACUUCUUUCcC 785 51734 98923.4uAuudTsdT 107274.1 uAAdTsdT HFE2 AD- 1273 A- uuAGGGAAAGAAGu 740 A-AAuAGACUuCUuUCcCu 785 51748 107281.6 CuAuUdTsdT 107277.3 AAdTsdT HFE2AD- 1273 A- uuAGGGAAAGAAGuc 740 A- AAuAGACUUCUUUCcC 785 51735 107280.3uAuUdTsdT 107274.2 uAAdTsdT HFE2 AD- 1273 A- uuAgGGAAAGAAGuC 740 A-AAuAGACUuCUuUCcCu 785 51749 107282.6 uAuUdTsdT 107277.4 AAdTsdT HFE2 AD-1273 A- uuAGGGAAAGAAGu 740 A- AAuAGACUuCUuUCcCu 785 51752 107281.7CuAuUdTsdT 107278.3 AadTsdT HFE2 AD- 1273 A- uuAGGGAAAGAAGuc 740 A-AAuAGACUUCUUUCCC 785 51738 98923.5 uAuudTsdT 107275.1 uAadTsdT HFE2 AD-1273 A- uuAGGGAAAGAAGuc 740 A- AAuAGACUUCUuUCCC 785 51730 98923.3uAuudTsdT 107273.1 uAAdTsdT HFE2 AD- 1273 A- uuAgGGAAAGAAGuC 740 A-AAuAGACUuCUuUCCC 785 51745 107282.5 uAuUdTsdT 107276.4 uAAdTsdT HFE2 AD-1273 A- uuAgGGAAAGAAGuC 740 A- AAuAGACUUCUUUCcC 785 51737 107282.3uAuUdTsdT 107274.4 uAAdTsdT HFE2 AD- 1273 A- uuAGGGAAAGAAGuc 740 A-AAuAGACUuCUuUCCC 785 51743 107280.5 uAuUdTsdT 107276.2 uAAdTsdT HFE2 AD-1273 A- uuAGGGAAAGAAGuc 740 A- AAuAGACUuCUuUCcCu 785 51751 107280.7uAuUdTsdT 107278.2 AadTsdT HFE2 AD- 1273 A- uuAGGGAAAGAAGuc 740 A-AAuAGACUuCUuUCcCu 785 51750 98923.8 uAuudTsdT 107278.1 AadTsdT HFE2 AD-1273 A- uuAgGGAAAGAAGuC 740 A- AAuAGACUUCUUUCCC 785 51741 107282.4uAuUdTsdT 107275.4 uAadTsdT HFE2 AD- 1273 A- uuAGGGAAAGAAGuc 740 A-AAuAGACUuCUuUCCC 785 51742 98923.6 uAuudTsdT 107276.1 uAAdTsdT HFE2 AD-1273 A- uuAgGGAAAGAAGuC 740 A- AAuAGACUUCUuUCCC 785 51733 107282.2uAuUdTsdT 107273.4 uAAdTsdT HFE2 AD- 1273 A- uuAGGGAAAGAAGuc 740 A-AAUAGACUuCUuUCcC 785 51755 107280.8 uAuUdTsdT 107279.2 uAadTsdT HFE2 AD-1273 A- uuAGGGAAAGAAGu 740 A- AAUAGACUuCUuUCcC 785 51756 107281.8CuAuUdTsdT 107279.3 uAadTsdT HFE2 AD- 1273 A- uuAGGGAAAGAAGu 740 A-AAuAGACUuCUUUCCC 785 51728 107281.1 CuAuUdTsdT 107272.3 uAAdTsdT HFE2AD- 1273 A- uuAgGGAAAGAAGuC 740 A- AAuAGACUuCUUUCCC 785 51729 107282.1uAuUdTsdT 107272.4 uAAdTsdT HFE2 AD- 1273 A- uuAGGGAAAGAAGuc 740 A-AAuAGACUuCUUUCCC 785 51726 98923.2 uAuudTsdT 107272.1 uAAdTsdT HFE2 AD-1273 A- uuAGGGAAAGAAGuc 740 A- AAuAGACUuCUuUCcCu 785 51746 98923.7uAuudTsdT 107277.1 AAdTsdT HFE2 AD- 1273 A- uuAgGGAAAGAAGuC 740 A-AAUAGACUuCUuUCcC 785 51757 107282.8 uAuUdTsdT 107279.4 uAadTsdT HFE2 AD-1273 A- uuAGGGAAAGAAGuc 740 A- AAuAGACUuCUUUCCC 785 51727 107280.1uAuUdTsdT 107272.2 uAAdTsdT HFE2 AD- 1273 A- uuAgGGAAAGAAGuC 740 A-AAuAGACUuCUuUCcCu 785 51753 107282.7 uAuUdTsdT 107278.4 AadTsdT HFE2 AD-1273 A- uuAGGGAAAGAAGuc 740 A- AAUAGACUuCUuUCcC 785 51754 98923.9uAuudTsdT 107279.1 uAadTsdT HFE2 AD- 1273 A- uuAGGGAAAGAAGuc 740 A-AAuAGACUUCUUUCCC 785 51739 107280.4 uAuUdTsdT 107275.2 uAadTsdT HFE2 AD-1274 A- uAGGGAAAGAAGucu 741 A- AAAuAGACUUCUUUC 786 47413 98925.1AuuudTsdT 98926.1 CCuAdTsdT HFE2 AD- 1279 A- AAAGAAGucuAuuuG 742 A-UcAUcAAAuAGACUUC 787 47419 98927.1 AuGAdTsdT 98928.1 UUUdTsdT HFE2 AD-1280 A- AAGAAGucuAuuuGA 743 A- UUcAUcAAAuAGACUU 788 47425 98929.1uGAAdTsdT 98930.1 CUUdTsdT HFE2 AD- 1303 A- uGuGuGuAAGGuAuG 744 A-AGAAcAuACCUuAcAcA 789 47431 98931.1 uucudTsdT 98932.1 cAdTsdT HFE2 AD-1366 A- GuGAAGGGAGucucu 745 A- AAGcAGAGACUCCCUU 790 47437 98933.1GcuudTsdT 98934.1 cACdTsdT HFE2 AD- 1367 A- uGAAGGGAGucucuGc 746 A-AAAGcAGAGACUCCCU 791 47396 98935.1 uuudTsdT 98936.1 UcAdTsdT HFE2 AD-1396 A- cAcAGGuAGGAcAGA 747 A- uACUUCUGUCCuACCU 792 47402 98937.1AGuAdTsdT 98938.1 GUGdTsdT HFE2 AD- 1397 A- AcAGGuAGGAcAGA 748 A-AuACUUCUGUCCuACC 793 47408 98939.1 AGuAudTsdT 98940.1 UGUdTsdT HFE2 AD-1399 A- AGGuAGGAcAGAAG 749 A- UGAuACUUCUGUCCuA 794 47414 98941.1uAucAdTsdT 98942.1 CCUdTsdT HFE2 AD- 1400 A- GGuAGGAcAGAAGu 750 A-AUGAuACUUCUGUCCu 795 47420 98943.1 AucAudTsdT 98944.1 ACCdTsdT HFE2 AD-1404 A- GGAcAGAAGuAucAu 751 A- AGGGAUGAuACUUCU 796 47426 98945.1cccudTsdT 98946.1 GUCCdTsdT HFE2 AD- 1441 A- uAuuAAAGcuAcAAAu 752 A-AGAAUUUGuAGCUUuA 797 47432 98947.1 ucudTsdT 98948.1 AuAdTsdT It shouldbe noted that unmodified versions of each of the modified sequencesshown are included within the scope of the invention.

TABLE 10B SEQ SEQ Start Sense ID Antisense ID Target Duplex ID PositionName Sense Sequence NO: Name Antisense Sequence NO: TFR2 AD-47814 64 A-uccAGAGAGcGcAAcAAcud 798 A- AGUUGUUGCGCUCUCUG 841 99594.1 TsdT 99595.1GAdTsdT TFR2 AD-47820 66 A- cAGAGAGcGcAAcAAcuGu 799 A-AcAGUUGUUGCGCUCUCU 842 99596.1 dTsdT 99597.1 GdTsdT TFR2 AD-47826 239 A-cAGGcAGccAAAccucAuud 35 A- AAUGAGGUUUGGCUGCC 38 99598.1 TsdT 99599.1UGdTsdT TFR2 AD-47819 772 A- AGcuGGuGuAcGcccAcuAd 800 A-uAGUGGGCGuAcACcAGCU 843 99674.1 TsdT 99675.1 dTsdT TFR2 AD-47832 884 A-ccAGAAGGuGAccAAuGcu 801 A- AGcAUUGGUcACCUUCUG 844 99600.1 dTsdT 99601.1GdTsdT TFR2 AD-47838 886 A- AGAAGGuGAccAAuGcucA 802 A-UGAGcAUUGGUcACCUUC 845 99602.1 dTsdT 99603.1 UdTsdT TFR2 AD-47844 915 A-GcucAAGGAGuGcucAuAud 803 A- AuAUGAGcACUCCUUGAG 846 99604.1 TsdT 99605.1CdTsdT TFR2 AD-47849 916 A- cucAAGGAGuGcucAuAuAd 804 A-uAuAUGAGcACUCCUUGA 847 99606.1 TsdT 99607.1 GdTsdT TFR2 AD-47854 920 A-AGGAGuGcucAuAuAcccAd 805 A- UGGGuAuAUGAGcACUCC 848 99608.1 TsdT 99609.1UdTsdT TFR2 AD-47815 922 A- GAGuGcucAuAuAcccAGAd 806 A-UCUGGGuAuAUGAGcACU 849 99610.1 TsdT 99611.1 CdTsdT TFR2 AD-47825 1004 A-AcAuGuGcAccuGGGAAcud 807 A- AGUUCCcAGGUGcAcAUG 850 99676.1 TsdT 99677.1UdTsdT TFR2 AD-47821 1048 A- cuuccuucAAucAAAcccAdTs 808 A-UGGGUUUGAUUGAAGGA 851 99612.1 dT 99613.1 AGdTsdT TFR2 AD-47827 1050 A-uccuucAAucAAAcccAGudT 809 A- ACUGGGUUUGAUUGAAG 852 99614.1 sdT 99615.1GAdTsdT TFR2 AD-47833 1051 A- ccuucAAucAAAcccAGuudT 47 A-AACUGGGUUUGAUUGAA 48 99616.1 sdT 99617.1 GGdTsdT TFR2 AD-51696 1051 A-ccuucAAucAAAcccAGuUdT 47 A- AACUGGGUUUGAuUGAA 48 107271.3 sdT 107257.2GGdTsdT TFR2 AD-51708 1051 A- ccuucAAucAAAcccAGuUdT 47 A-AACUGGGUuUGAuUGAAG 48 107271.5 sdT 107259.2 GdTsdT TFR2 AD-51700 1051 A-ccuucAAucAAAcccAGuudT 47 A- AACUGGGUuUGAUUGAA 48 99616.5 sdT 107258.1GGdTsdT TFR2 AD-51701 1051 A- ccuucAAucAAAcccAGuudT 47 A-AACuGGGUuUGAUUGAAG 48 99616.13 sdT 107266.1 GdTsdT TFR2 AD-51702 1051 A-ccuucAAucAAAcccAGuUdT 47 A- AACUGGGUuUGAUUGAA 48 107271.4 sdT 107258.2GGdTsdT TFR2 AD-51707 1051 A- ccuucAAucAAAcccAGuudT 47 A-AACuGGGUuUGAuUGAAG 48 99616.14 sdT 107267.1 GdTsdT TFR2 AD-51694 1051 A-ccuucAAucAAAcccAGuudT 47 A- AACUGGGUUUGAuUGAA 48 99616.4 sdT 107257.1GGdTsdT TFR2 AD-51706 1051 A- ccuucAAucAAAcccAGuudT 47 A-AACUGGGUuUGAuUGAAG 48 99616.6 sdT 107259.1 GdTsdT TFR2 AD-51695 1051 A-ccuucAAucAAAcccAGuudT 47 A- AACuGGGUUUGAuUGAAG 48 99616.12 sdT 107265.1GdTsdT TFR2 AD-51713 1051 A- ccuucAAucAAAcccAGuudT 47 A-AACuGGGUuUGAuuGAAG 48 99616.15 sdT 107268.1 GdTsdT TFR2 AD-51714 1051 A-ccuucAAucAAAcccAGuUdT 47 A- AACUGGGUuUGAuuGAAG 48 107271.6 sdT 107260.2GdTsdT TFR2 AD-51683 1051 A- ccuucAAucAAAcccAGuudT 47 A-AACuGGGUUUGAUUGAA 48 99616.10 sdT 107263.1 GgdTsdT TFR2 AD-51712 1051 A-ccuucAAucAAAcccAGuudT 47 A- AACUGGGUuUGAuuGAAG 48 99616.7 sdT 107260.1GdTsdT TFR2 AD-51720 1051 A- ccuucAAucAAAcccAGuUdT 47 A-AACUGGGUuUGAuuGAAG 48 107271.7 sdT 107261.2 gdTsdT TFR2 AD-51719 1051 A-ccuucAAucAAAcccAGuudT 47 A- AACuGGGUuUGAuuGAAGg 48 99616.16 sdT 107269.1dTsdT TFR2 AD-51684 1051 A- ccuucAAucAAAcccAGuUdT 47 A-AACUGGGUUUGAUUGAA 48 107271.1 sdT 107255.2 GgdTsdT TFR2 AD-51690 1051 A-ccuucAAucAAAcccAGuUdT 47 A- AACUGGGUUUGAUuGAA 48 107271.2 sdT 107256.2GGdTsdT TFR2 AD-51689 1051 A- ccuucAAucAAAcccAGuudT 47 A-AACuGGGUUUGAUuGAAG 48 99616.11 sdT 107264.1 GdTsdT TFR2 AD-51682 1051 A-ccuucAAucAAAcccAGuudT 47 A- AACUGGGUUUGAUUGAA 48 99616.2 sdT 107255.1GgdTsdT TFR2 AD-51688 1051 A- ccuucAAucAAAcccAGuudT 47 A-AACUGGGUUUGAUuGAA 48 99616.3 sdT 107256.1 GGdTsdT TFR2 AD-51718 1051 A-ccuucAAucAAAcccAGuudT 47 A- AACUGGGUuUGAuuGAAG 48 99616.8 sdT 107261.1gdTsdT TFR2 AD-51725 1051 A- ccuucAAucAAAcccAGuudT 47 A-AACuGGGUuUGAUuGAAG 48 99616.17 sdT 107270.1 gdTsdT TFR2 AD-51724 1051 A-ccuucAAucAAAcccAGuudT 47 A- AACUGGGUuUGAUuGAAG 48 99616.9 sdT 107262.1gdTsdT TFR2 AD-47839 1067 A- GuucccuccAGuuGcAucAdTs 810 A-UGAUGcAACUGGAGGGAA 853 99618.1 dT 99619.1 CdTsdT TFR2 AD-47845 1068 A-uucccuccAGuuGcAucAudTs 811 A- AUGAUGcAACUGGAGGGA 854 99620.1 dT 99621.1AdTsdT TFR2 AD-47850 1299 A- cGcucAGAGccAGAucAcud 812 A-AGUGAUCUGGCUCUGAG 855 99622.1 TsdT 99623.1 CGdTsdT TFR2 AD-47855 1355 A-AGGAGcAGcuAAAuccGcu 813 A- AGCGGAUUuAGCUGCUCC 856 99624.1 dTsdT 99625.1UdTsdT TFR2 AD-47816 1441 A- cccGcAGAAGucuccucuudTs 814 A-AAGAGGAGACUUCUGCG 857 99626.1 dT 99627.1 GGdTsdT TFR2 AD-47831 1548 A-GuGuAcGuGAGccuGGAcA 815 A- UGUCcAGGCUcACGuAcAC 858 99678.1 dTsdT 99679.1dTsdT TFR2 AD-47822 1584 A- GAcAAGuuucAuGccAAGA 816 A-UCUUGGcAUGAAACUUGU 859 99628.1 dTsdT 99629.1 CdTsdT TFR2 AD-47828 1612A- uucuGAcAAGucucAuuGAd 817 A- UcAAUGAGACUUGUcAGA 860 99630.1 TsdT99631.1 AdTsdT TFR2 AD-47834 1614 A- cuGAcAAGucucAuuGAGAd 818 A-UCUcAAUGAGACUUGUcA 861 99632.1 TsdT 99633.1 GdTsdT TFR2 AD-47840 1616 A-GAcAAGucucAuuGAGAGu 819 A- ACUCUcAAUGAGACUUGU 862 99634.1 dTsdT 99635.1CdTsdT TFR2 AD-47846 1618 A- cAAGucucAuuGAGAGuGud 820 A-AcACUCUcAAUGAGACUU 863 99636.1 TsdT 99637.1 GdTsdT TFR2 AD-47851 2140 A-AGcGAcuGAcAcGcAuGuA 821 A- uAcAUGCGUGUcAGUCGC 864 99638.1 dTsdT 99639.1UdTsdT TFR2 AD-47856 2142 A- cGAcuGAcAcGcAuGuAcAd 822 A-UGuAcAUGCGUGUcAGUC 865 99640.1 TsdT 99641.1 GdTsdT TFR2 AD-47817 2143 A-GAcuGAcAcGcAuGuAcAA 823 A- UUGuAcAUGCGUGUcAGU 866 99642.1 dTsdT 99643.1CdTsdT TFR2 AD-47823 2146 A- uGAcAcGcAuGuAcAAcGud 824 A-ACGUUGuAcAUGCGUGUc 867 99644.1 TsdT 99645.1 AdTsdT TFR2 AD-47837 2151 A-cGcAuGuAcAAcGuGcGcAd 825 A- UGCGcACGUUGuAcAUGC 868 99680.1 TsdT 99681.1GdTsdT TFR2 AD-47843 2152 A- GcAuGuAcAAcGuGcGcAud 826 A-AUGCGcACGUUGuAcAUG 869 99682.1 TsdT 99683.1 CdTsdT TFR2 AD-47829 2154 A-AuGuAcAAcGuGcGcAuAA 827 A- UuAUGCGcACGUUGuAcAU 870 99646.1 dTsdT 99647.1dTsdT TFR2 AD-47835 2155 A- uGuAcAAcGuGcGcAuAAud 828 A-AUuAUGCGcACGUUGuAcA 871 99648.1 TsdT 99649.1 dTsdT TFR2 AD-47841 2170 A-uAAuGcGGGuGGAGuucuA 829 A- uAGAACUCcACCCGcAUuA 872 99650.1 dTsdT 99651.1dTsdT TFR2 AD-51703 2170 A- uAAuGcGGGuGGAGuucuA 829 A-UAGAACuCcACCCGcAUuA 872 99650.6 dTsdT 107249.1 dTsdT TFR2 AD-51710 2170A- uAAuGcGGGuGGAGuUCu 829 A- UAGAACUCcACCCGcAuuA 872 107254.2 AdTsdT107246.3 dTsdT TFR2 AD-51697 2170 A- uAAuGcGGGuGGAGuucuA 829 A-UAGAACUCcACCCGcAuuad 872 99650.5 dTsdT 107248.1 TsdT TFR2 AD-51692 2170A- uAAuGcGGGuGGAGuuCuA 829 A- UAGAACUCcACCCGcAUua 872 107253.3 dTsdT107247.2 dTsdT TFR2 AD-51685 2170 A- uAAuGcGGGuGGAGuucuA 829 A-UAGAACUCcACCCGcAuuA 872 99650.3 dTsdT 107246.1 dTsdT TFR2 AD-51691 2170A- uAAuGcGGGuGGAGuucuA 829 A- UAGAACUCcACCCGcAUua 872 99650.4 dTsdT107247.1 dTsdT TFR2 AD-51698 2170 A- uAAuGcGGGuGGAGuuCuA 829 A-UAGAACUCcACCCGcAuuad 872 107253.4 dTsdT 107248.2 TsdT TFR2 AD-51686 2170A- uAAuGcGGGuGGAGuuCuA 829 A- UAGAACUCcACCCGcAuuA 872 107253.2 dTsdT107246.2 dTsdT TFR2 AD-51709 2170 A- uAAuGcGGGuGGAGuucuA 829 A-UAGAACuCcACCCGcAuuA 872 99650.7 dTsdT 107250.1 dTsdT TFR2 AD-51679 2170A- uAAuGcGGGuGGAGuucuA 829 A- UAGAACUCcACCCGcAUuA 872 99650.2 dTsdT107245.1 dTsdT TFR2 AD-51705 2170 A- uAAuGcGGGuGGAGuUCu 829 A-UAGAACuCcACCCGcAUuA 872 107254.5 AdTsdT 107249.3 dTsdT TFR2 AD-517042170 A- uAAuGcGGGuGGAGuUCu 829 A- UAGAACUCcACCCGcAUuA 872 107254.1AdTsdT 107245.3 dTsdT TFR2 AD-51687 2170 A- uAAuGcGGGuGGAGuuCuA 829 A-UAGAACuCcACCCGcAuuA 872 107253.6 dTsdT 107250.2 dTsdT TFR2 AD-51681 2170A- uAAuGcGGGuGGAGuuCuA 829 A- UAGAACuCcACCCGcAUuA 872 107253.5 dTsdT107249.2 dTsdT TFR2 AD-51716 2170 A- uAAuGcGGGuGGAGuUCu 829 A-UAGAACUCcACCCGcAUua 872 107254.3 AdTsdT 107247.3 dTsdT TFR2 AD-516932170 A- uAAuGcGGGuGGAGuuCuA 829 A- UAGAACuCcACCCGcAUuad 872 107253.7dTsdT 107251.2 TsdT TFR2 AD-51711 2170 A- uAAuGcGGGuGGAGuUCu 829 A-UAGAACuCcACCCGcAuuA 872 107254.6 AdTsdT 107250.3 dTsdT TFR2 AD-516992170 A- uAAuGcGGGuGGAGuuCuA 829 A- UAGAACuCcACCCGcAuuad 872 107253.8dTsdT 107252.2 TsdT TFR2 AD-51722 2170 A- uAAuGcGGGuGGAGuUCu 829 A-UAGAACUCcACCCGcAuuad 872 107254.4 AdTsdT 107248.3 TsdT TFR2 AD-517152170 A- uAAuGcGGGuGGAGuucuA 829 A- UAGAACuCcACCCGcAUuad 872 99650.8dTsdT 107251.1 TsdT TFR2 AD-51680 2170 A- uAAuGcGGGuGGAGuuCuA 829 A-UAGAACUCcACCCGcAUuA 872 107253.1 dTsdT 107245.2 dTsdT TFR2 AD-51717 2170A- uAAuGcGGGuGGAGuUCu 829 A- UAGAACuCcACCCGcAUuad 872 107254.7 AdTsdT107251.3 TsdT TFR2 AD-51723 2170 A- uAAuGcGGGuGGAGuUCu 829 A-UAGAACuCcACCCGcAuuad 872 107254.8 AdTsdT 107252.3 TsdT TFR2 AD-517212170 A- uAAuGcGGGuGGAGuucuA 829 A- UAGAACuCcACCCGcAuuad 872 99650.9dTsdT 107252.1 TsdT TFR2 AD-47847 2178 A- GuGGAGuucuAcuuccuuudTs 830 A-AAAGGAAGuAGAACUCcA 873 99652.1 dT 99653.1 CdTsdT TFR2 AD-47852 2224 A-cGuuccGccAcAucuucAudTs 831 A- AUGAAGAUGUGGCGGAA 874 99654.1 dT 99655.1CGdTsdT TFR2 AD-47857 2425 A- GGAAcAuuGAuAAcAAcuu 832 A-AAGUUGUuAUcAAUGUUC 875 99656.1 dTsdT 99657.1 CdTsdT TFR2 AD-47818 2602A- cAGcAcAGAuAuccAcAcAd 833 A- UGUGUGGAuAUCUGUGCU 876 99658.1 TsdT99659.1 GdTsdT TFR2 AD-47824 2656 A- GGucAuAcuGucGGuuAAud 834 A-AUuAACCGAcAGuAUGAC 877 99660.1 TsdT 99661.1 CdTsdT TFR2 AD-47830 2658 A-ucAuAcuGucGGuuAAucAd 835 A- UGAUuAACCGAcAGuAUG 878 99662.1 TsdT 99663.1AdTsdT TFR2 AD-47836 2660 A- AuAcuGucGGuuAAucAGAd 836 A-UCUGAUuAACCGAcAGuA 879 99664.1 TsdT 99665.1 UdTsdT TFR2 AD-47842 2662 A-AcuGucGGuuAAucAGAGA 837 A- UCUCUGAUuAACCGAcAG 880 99666.1 dTsdT 99667.1UdTsdT TFR2 AD-47848 2719 A- GGuccuccAuAccuAGAGAd 838 A-UCUCuAGGuAUGGAGGAC 881 99668.1 TsdT 99669.1 CdTsdT TFR2 AD-47853 2795 A-ucGcuGGcAccAuAGccuudT 839 A- AAGGCuAUGGUGCcAGCG 882 99670.1 sdT 99671.1AdTsdT TFR2 AD-47858 2802 A- cAccAuAGccuuAuGGccAdT 840 A-UGGCcAuAAGGCuAUGGU 883 99672.1 sdT 99673.1 GdTsdT It should be notedthat unmodified versions of each of the modified sequences shown areincluded within the scope of the invention.

TABLE 11 Secondary Target single-dose Table 11 Start Duplex Posi- 10 nMoverall 0.1 nM overall 0.01 nM overall Target Reactivity Name tion AvgSD Avg SD Avg SD HFE2 Human AD-47391 177 97.5 10.8 111.9 21.2 HFE2 HumanAD-47397 193 27.3 4.2 36.9 3.3 HFE2 Human AD-47403 195 31.2 10.0 48.67.6 HFE2 Human AD-47409 199 82.3 15.8 89.5 11.4 HFE2 Human AD-47415 20044.8 5.9 51.1 7.0 HFE2 Human AD-47421 206 27.8 8.1 28.8 0.9 HFE2 HumanAD-47427 211 96.4 25.8 79.8 18.2 HFE2 Human AD-47433 244 7.5 1.3 21.24.0 HFE2 Human AD-47392 257 8.0 2.0 20.5 8.1 HFE2 Human AD-47398 26130.0 6.5 45.9 6.4 HFE2 Human AD-47404 290 9.3 2.8 20.5 0.5 HFE2 HumanAD-47410 360 28.7 9.8 36.7 1.8 HFE2 Human AD-47416 367 72.3 12.5 79.219.3 HFE2 Human AD-47422 404 20.4 2.5 35.4 2.5 HFE2 Human AD-47428 41566.8 22.5 80.6 11.4 HFE2 Human AD-47434 417 34.7 5.9 28.6 3.4 HFE2 HumanAD-47393 472 96.3 9.7 99.8 31.3 HFE2 Human AD-47399 585 10.0 6.6 16.30.6 HFE2 Human AD-47405 587 11.3 2.1 14.0 0.4 HFE2 Human AD-47417 63839.3 2.0 62.6 7.6 HFE2 Human AD-47423 642 109.4 4.1 58.5 0.9 HFE2 HumanAD-47429 646 56.0 13.0 76.3 21.8 HFE2 Human AD-47435 656 17.7 1.4 29.39.4 HFE2 Human AD-47394 657 8.8 7.3 9.8 6.3 HFE2 Human AD-47400 678 21.22.8 25.1 8.4 HFE2 Human AD-47406 1121 12.9 1.4 20.5 1.3 HFE2 HumanAD-47412 1151 16.5 5.2 11.8 3.2 HFE2 Human AD-47418 1152 16.0 1.6 8.42.2 HFE2 Human AD-47424 1203 9.2 1.6 14.0 2.4 HFE2 Human AD-47430 122814.8 2.7 19.2 0.9 HFE2 Human AD-47436 1230 17.9 9.6 19.7 1.4 HFE2 HumanAD-47395 1233 15.3 2.1 12.7 2.2 HFE2 Human AD-47401 1272 6.3 1.2 10.50.9 HFE2 Human AD-47407 1273 5.6 1.8 5.6 0.8 HFE2 Human AD-51740 12735.7 0.0 6.5 0.7 6.3 0.4 HFE2 Human AD-51747 1273 7.1 1.6 6.0 0.1 7.0 0.1HFE2 Human AD-51744 1273 11.8 5.8 18.4 14.1 7.7 0.0 HFE2 Human AD-517311273 6.2 0.7 7.1 0.2 8.1 3.2 HFE2 Human AD-51736 1273 6.3 0.3 7.2 0.78.2 0.5 HFE2 Human AD-51732 1273 6.0 1.0 8.2 0.5 8.3 0.6 HFE2 HumanAD-51734 1273 6.9 0.3 14.5 13.3 8.4 1.4 HFE2 Human AD-51748 1273 6.6 0.27.7 0.9 8.5 1.3 HFE2 Human AD-51735 1273 6.4 1.5 6.3 0.3 8.5 0.7 HFE2Human AD-51749 1273 6.8 1.0 8.3 0.4 8.7 2.2 HFE2 Human AD-51752 127312.7 6.4 10.3 3.6 8.8 1.0 HFE2 Human AD-51738 1273 5.8 0.6 9.2 3.0 8.91.4 HFE2 Human AD-51730 1273 7.6 1.7 7.8 1.0 9.3 0.5 HFE2 Human AD-517451273 5.8 0.4 6.5 1.6 9.5 1.2 HFE2 Human AD-51737 1273 5.9 0.1 19.8 18.49.6 1.3 HFE2 Human AD-51743 1273 6.5 1.6 7.0 1.5 9.9 2.0 HFE2 HumanAD-51751 1273 6.4 1.4 7.5 1.6 10.3 1.6 HFE2 Human AD-51750 1273 6.9 0.28.8 0.3 10.7 1.0 HFE2 Human AD-51741 1273 6.0 2.1 8.5 1.1 10.8 4.0 HFE2Human AD-51742 1273 7.0 1.0 6.1 0.2 11.0 0.9 HFE2 Human AD-51733 12736.7 1.1 7.2 0.1 11.0 1.3 HFE2 Human AD-51755 1273 6.1 0.8 13.4 6.9 11.22.8 HFE2 Human AD-51756 1273 9.8 0.3 8.9 0.4 11.6 0.3 HFE2 HumanAD-51728 1273 7.1 0.8 8.2 0.2 11.6 0.6 HFE2 Human AD-51729 1273 6.8 1.28.9 0.4 11.7 0.5 HFE2 Human AD-51726 1273 7.1 0.6 9.0 1.0 12.6 2.4 HFE2Human AD-51746 1273 7.3 1.4 14.9 6.0 12.6 5.5 HFE2 Human AD-51757 12739.1 2.0 10.4 0.6 13.1 1.9 HFE2 Human AD-51727 1273 6.7 1.0 8.5 1.1 13.80.4 HFE2 Human AD-51753 1273 7.2 0.3 13.6 8.4 14.2 7.8 HFE2 HumanAD-51754 1273 6.9 0.4 10.1 1.0 14.7 2.8 HFE2 Human AD-51739 1273 6.1 0.18.2 0.1 14.8 8.9 HFE2 Human AD-47413 1274 7.2 0.2 6.4 0.9 HFE2 HumanAD-47419 1279 8.6 2.3 10.0 2.2 HFE2 Human AD-47425 1280 14.5 1.0 14.10.8 HFE2 Human AD-47431 1303 49.5 0.6 72.2 0.7 HFE2 Human AD-47437 13666.4 4.2 11.4 2.4 HFE2 Human AD-47396 1367 4.6 0.1 10.0 0.2 HFE2 HumanAD-47402 1396 11.8 0.2 19.9 4.4 HFE2 Human AD-47408 1397 12.0 3.4 13.70.2 HFE2 Human AD-47414 1399 5.6 1.5 8.2 0.1 HFE2 Human AD-47420 14003.6 1.0 5.7 0.8 HFE2 Human AD-47426 1404 13.7 3.8 27.1 3.1 HFE2 HumanAD-47432 1441 3.8 0.0 5.6 1.0 TFR2 Human AD-47814 64 7.8 0.4 16.3 0.1TFR2 Human AD-47820 66 13.7 2.5 25.1 3.7 TFR2 Human AD-47826 239 13.51.8 25.4 4.3 TFR2 Human AD-47819 772 112.4 2.9 102.9 3.8 TFR2 HumanAD-47832 884 24.2 1.8 52.4 2.7 TFR2 Human AD-47838 886 23.6 0.4 39.0 1.6TFR2 Human AD-47844 915 19.5 3.9 40.9 4.5 TFR2 Human AD-47849 916 14.26.9 22.8 0.5 TFR2 Human AD-47854 920 69.4 4.2 88.3 0.8 TFR2 HumanAD-47815 922 66.3 6.7 71.2 8.8 TFR2 Human AD-47825 1004 23.9 2.9 46.23.8 TFR2 Human AD-47821 1048 57.4 15.9 78.5 5.0 TFR2 Human AD-47827 105018.9 8.3 37.9 2.9 TFR2 Human AD-47833 1051 8.3 4.3 19.7 5.4 TFR2 HumanAD-51696 1051 8.0 2.1 21.1 2.1 27.2 0.5 TFR2 Human AD-51708 1051 8.8 1.217.7 0.8 28.5 3.7 TFR2 Human AD-51700 1051 9.3 1.2 19.8 3.7 30.1 5.0TFR2 Human AD-51701 1051 9.4 0.6 22.3 8.1 30.8 2.7 TFR2 Human AD-517021051 8.7 2.1 19.7 0.1 30.9 1.4 TFR2 Human AD-51707 1051 8.1 2.5 19.1 2.632.2 8.2 TFR2 Human AD-51694 1051 9.3 1.9 19.3 2.5 38.8 0.0 TFR2 HumanAD-51706 1051 8.4 0.3 19.5 1.5 39.9 6.9 TFR2 Human AD-51695 1051 10.11.6 19.9 2.4 40.1 4.2 TFR2 Human AD-51713 1051 14.6 1.8 45.3 2.5 59.01.6 TFR2 Human AD-51714 1051 22.1 0.1 44.2 1.5 62.8 1.7 TFR2 HumanAD-51683 1051 9.7 0.6 36.5 2.4 66.0 2.4 TFR2 Human AD-51712 1051 21.22.6 44.1 4.5 67.1 5.9 TFR2 Human AD-51720 1051 34.5 6.1 58.3 10.4 67.40.0 TFR2 Human AD-51719 1051 38.6 1.2 57.7 1.2 68.8 3.2 TFR2 HumanAD-51684 1051 14.7 3.5 48.3 1.9 69.3 3.5 TFR2 Human AD-51690 1051 19.70.0 49.8 0.5 74.1 12.6 TFR2 Human AD-51689 1051 40.5 2.4 53.1 9.7 75.06.4 TFR2 Human AD-51682 1051 12.7 1.1 42.3 10.1 75.7 6.0 TFR2 HumanAD-51688 1051 34.9 2.9 62.2 6.9 78.1 3.2 TFR2 Human AD-51718 1051 31.66.6 53.1 6.5 80.2 1.3 TFR2 Human AD-51725 1051 47.9 5.0 76.1 1.8 83.73.2 TFR2 Human AD-51724 1051 52.0 1.2 66.1 32.9 87.8 14.9 TFR2 HumanAD-47839 1067 54.0 3.1 71.5 8.4 TFR2 Human AD-47845 1068 105.7 20.1 98.03.0 TFR2 Human AD-47850 1299 16.7 4.8 21.3 3.2 TFR2 Human AD-47855 135564.6 0.5 66.1 8.0 TFR2 Human AD-47816 1441 10.6 2.6 30.6 6.9 TFR2 HumanAD-47831 1548 22.8 0.2 36.6 9.5 TFR2 Human AD-47822 1584 57.2 7.0 72.61.6 TFR2 Human AD-47828 1612 38.2 5.9 61.2 9.9 TFR2 Human AD-47834 16149.2 3.6 20.1 3.0 TFR2 Human AD-47840 1616 50.1 3.7 55.6 3.8 TFR2 HumanAD-47846 1618 75.0 7.9 94.6 4.3 TFR2 Human AD-47851 2140 94.1 0.4 101.310.6 TFR2 Human AD-47856 2142 63.3 4.1 60.7 3.1 TFR2 Human AD-47817 214350.2 2.7 50.3 6.5 TFR2 Human AD-47823 2146 26.1 2.3 40.9 3.3 TFR2 HumanAD-47837 2151 119.5 21.7 89.5 6.9 TFR2 Human AD-47843 2152 20.6 1.7 34.97.8 TFR2 Human AD-47829 2154 53.4 4.1 60.3 0.5 TFR2 Human AD-47835 215515.5 1.8 18.3 2.4 TFR2 Human AD-47841 2170 26.6 1.5 24.7 2.0 TFR2 HumanAD-51703 2170 25.2 2.8 27.9 3.5 23.2 1.1 TFR2 Human AD-51710 2170 22.13.4 23.1 0.5 24.0 0.6 TFR2 Human AD-51697 2170 30.9 3.6 25.3 0.9 24.50.8 TFR2 Human AD-51692 2170 23.1 1.3 24.6 1.2 24.9 6.4 TFR2 HumanAD-51685 2170 24.6 2.2 23.9 0.6 25.6 1.7 TFR2 Human AD-51691 2170 29.13.2 21.3 0.2 26.4 3.7 TFR2 Human AD-51698 2170 23.1 2.3 25.8 3.0 26.82.8 TFR2 Human AD-51686 2170 20.7 2.5 24.7 0.7 27.5 1.4 TFR2 HumanAD-51709 2170 23.1 1.3 25.1 2.7 27.7 2.1 TFR2 Human AD-51679 2170 27.42.2 26.4 4.3 28.3 5.1 TFR2 Human AD-51705 2170 27.8 5.3 24.6 2.0 28.82.4 TFR2 Human AD-51704 2170 23.9 2.1 26.1 0.5 29.2 4.6 TFR2 HumanAD-51687 2170 20.8 3.9 27.7 2.8 29.4 1.0 TFR2 Human AD-51681 2170 30.01.8 31.2 1.8 29.5 4.7 TFR2 Human AD-51716 2170 20.0 1.7 25.9 2.2 30.21.1 TFR2 Human AD-51693 2170 26.2 0.8 26.1 1.0 30.6 0.6 TFR2 HumanAD-51711 2170 20.8 0.5 24.8 3.2 31.3 3.0 TFR2 Human AD-51699 2170 20.90.7 27.3 1.5 31.7 5.1 TFR2 Human AD-51722 2170 28.3 3.7 30.0 0.5 32.11.2 TFR2 Human AD-51715 2170 22.2 6.1 30.4 0.6 34.6 1.3 TFR2 HumanAD-51680 2170 26.4 2.5 26.7 5.4 36.6 2.6 TFR2 Human AD-51717 2170 28.26.2 24.6 0.2 37.2 7.7 TFR2 Human AD-51723 2170 25.9 4.0 30.7 4.0 40.73.1 TFR2 Human AD-51721 2170 30.7 1.6 28.1 0.9 40.8 0.3 TFR2 HumanAD-47847 2178 21.7 2.1 25.1 3.5 TFR2 Human AD-47852 2224 71.4 2.2 66.77.1 TFR2 Human AD-47857 2425 37.4 4.8 29.5 5.4 TFR2 Human AD-47818 260248.3 4.8 50.8 4.3 TFR2 Human AD-47824 2656 19.9 3.3 25.7 0.1 TFR2 HumanAD-47830 2658 25.8 7.7 25.8 6.4 TFR2 Human AD-47836 2660 34.6 0.1 37.46.1 TFR2 Human AD-47842 2662 19.2 6.8 26.3 1.1 TFR2 Human AD-47848 271976.8 2.2 90.1 9.7 TFR2 Human AD-47853 2795 28.1 6.3 43.7 3.8 TFR2 HumanAD-47858 2802 66.9 8.2 73.6 3.4 Data are expressed as percent of control(Mock transfected or 1955).

TABLE 12 Secondary Target dose-response Table 12 Target ReactivityDuplex Name Start Position IC50 (nM) HFE2 HumaWn AD-47394 657 0.004 HFE2Human AD-47395 1233 0.011 HFE2 Human AD-47407 1273 0.002 HFE2 HumanAD-51747 1273 0.001 HFE2 Human AD-51736 1273 0.001 HFE2 Human AD-517341273 0.001 HFE2 Human AD-51732 1273 0.002 HFE2 Human AD-51731 1273 0.002HFE2 Human AD-51744 1273 0.002 HFE2 Human AD-51748 1273 0.002 HFE2 HumanAD-51735 1273 0.002 HFE2 Human AD-47407 1273 0.002 HFE2 Human AD-517401273 0.003 HFE2 Human AD-47413 1274 0.003 HFE2 Human AD-47425 1280 0.021HFE2 Human AD-47437 1366 0.015 HFE2 Human AD-47396 1367 0.013 HFE2 HumanAD-47414 1399 0.005 HFE2 Human AD-47420 1400 0.010 HFE2 Human AD-474321441 0.004 TFR2 Human AD-47814 64 0.012 TFR2 Human AD-47820 66 0.011TFR2 Human AD-47826 239 0.014 TFR2 Human AD-47849 916 0.067 TFR2 HumanAD-47833 1051 0.013 TFR2 Human AD-51701 1051 0.015 TFR2 Human AD-517081051 0.017 TFR2 Human AD-51700 1051 0.017 TFR2 Human AD-47833 1051 0.023TFR2 Human AD-51696 1051 0.024 TFR2 Human AD-47850 1299 0.011 TFR2 HumanAD-47834 1614 0.014 TFR2 Human AD-47835 2155 0.023 TFR2 Human AD-478412170 0.009 TFR2 Human AD-51710 2170 0.003 TFR2 Human AD-51703 2170 0.005TFR2 Human AD-51697 2170 0.006 TFR2 Human AD-51692 2170 0.010 TFR2 HumanAD-47841 2170 0.024 TFR2 Human AD-47847 2178 0.013

TABLE 13 TFR2 Duplex Sequences Table SEQ SEQ 13 Start Sense ID AntisenseID Target Duplex ID Position Name Sense Sequence NO: Name AntisenseSequence NO: TFR2 AD-52549 64 A- uccAGAGAGcGcAAcAAcUdTsdT 798 A-108798.2AGUUGUUGCGCUCUCuGGAdTsdT 841 108802.1 TFR2 AD-52550 64 A-uccAGAGAGcGcAAcAAcUdTsdT 798 A-108803.2 AGUUGUUGCGCUCUCuGGadTsdT 841108802.5 TFR2 AD-52555 64 A- uccAGAGAGcGcAAcAAcUdTsdT 798 A-108799.2AGUUGUUGCGCUCuCuGGAdTsdT 841 108802.2 TFR2 AD-52556 64 A-uccAGAGAGcGcAAcAAcUdTsdT 798 A-108804.2 AGUUGUUGCGCUCuCuGGadTsdT 841108802.6 TFR2 AD-52561 64 A- uccAGAGAGcGcAAcAAcUdTsdT 798 A-108800.2AGUUGUUGCGCuCuCuGGAdTsdT 841 108802.3 TFR2 AD-52562 64 A-uccAGAGAGcGcAAcAAcUdTsdT 798 A-108805.2 AGUUGUUGCGCuCuCuGGadTsdT 841108802.7 TFR2 AD-52567 64 A- uccAGAGAGcGcAAcAAcUdTsdT 798 A-108801.2AGUUGuUGCGCuCuCuGGAdTsdT 841 108802.4 TFR2 AD-52568 64 A-uccAGAGAGcGcAAcAAcUdTsdT 798 A-108806.2 AGUUGuUGCGCuCuCuGGadTsdT 841108802.8 TFR2 AD-52572 64 A-99594.2 uccAGAGAGcGcAAcAAcudTsdT 798A-108798.1 AGUUGUUGCGCUCUCuGGAdTsdT 841 TFR2 AD-52573 64 A-99594.6uccAGAGAGcGcAAcAAcudTsdT 798 A-108803.1 AGUUGUUGCGCUCUCuGGadTsdT 841TFR2 AD-52577 64 A-99594.3 uccAGAGAGcGcAAcAAcudTsdT 798 A-108799.1AGUUGUUGCGCUCuCuGGAdTsdT 841 TFR2 AD-52578 64 A-99594.7uccAGAGAGcGcAAcAAcudTsdT 798 A-108804.1 AGUUGUUGCGCUCuCuGGadTsdT 841TFR2 AD-52582 64 A-99594.4 uccAGAGAGcGcAAcAAcudTsdT 798 A-108800.1AGUUGUUGCGCuCuCuGGAdTsdT 841 TFR2 AD-52583 64 A-99594.8uccAGAGAGcGcAAcAAcudTsdT 798 A-108805.1 AGUUGUUGCGCuCuCuGGadTsdT 841TFR2 AD-52587 64 A-99594.5 uccAGAGAGcGcAAcAAcudTsdT 798 A-108801.1AGUUGuUGCGCuCuCuGGAdTsdT 841 TFR2 AD-52588 64 A-99594.9uccAGAGAGcGcAAcAAcudTsdT 798 A-108806.1 AGUUGuUGCGCuCuCuGGadTsdT 841TFR2 AD-52551 239 A- cAGGcAGccAAAccucAuUdTsdT 35 A-108810.2AAUGAGGUuUGGCuGcCuGdTsdT 38 108811.1 TFR2 AD-52552 239 A-cAGGcAGccAAAccucAuUdTsdT 35 A-108816.1 AAuGAGGUuUGGCUGcCugdTsdT 38108811.3 TFR2 AD-52557 239 A- cAGGcAGccAAAccuCAuUdTsdT 35 A-108810.3AAUGAGGUuUGGCuGcCuGdTsdT 38 108812.1 TFR2 AD-52558 239 A-cAGGcAGccAAAccuCAuUdTsdT 35 A-108816.2 AAuGAGGUuUGGCUGcCugdTsdT 38108812.3 TFR2 AD-52563 239 A- cAGGcAGccAAAcCuCAuUdTsdT 35 A-108810.4AAUGAGGUuUGGCuGcCuGdTsdT 38 108813.1 TFR2 AD-52564 239 A-cAGGcAGccAAAcCuCAuUdTsdT 35 A-108816.3 AAuGAGGUuUGGCUGcCugdTsdT 38108813.3 TFR2 AD-52569 239 A- cAGGcAGcCAAAcCuCAuUdTsdT 35 A-108810.5AAUGAGGUuUGGCuGcCuGdTsdT 38 108814.1 TFR2 AD-52570 239 A-cAGGcAGcCAAAcCuCAuUdTsdT 35 A-108816.4 AAuGAGGUuUGGCUGcCugdTsdT 38108814.3 TFR2 AD-52574 239 A-99598.2 cAGGcAGccAAAccucAuudTsdT 35A-108807.1 AAUGAGGUUUGGCUGCCuGdTsdT 38 TFR2 AD-52575 239 A-cAGGcAGccAAAccucAuUdTsdT 35 A-108815.1 AAUGAGGUuUGGCUGcCugdTsdT 38108811.2 TFR2 AD-52579 239 A-99598.3 cAGGcAGccAAAccucAuudTsdT 35A-108808.1 AAUGAGGUUUGGCUGcCuGdTsdT 38 TFR2 AD-52580 239 A-cAGGcAGccAAAccuCAuUdTsdT 35 A-108815.2 AAUGAGGUuUGGCUGcCugdTsdT 38108812.2 TFR2 AD-52584 239 A-99598.4 cAGGcAGccAAAccucAuudTsdT 35A-108809.1 AAUGAGGUUUGGCuGcCuGdTsdT 38 TFR2 AD-52585 239 A-cAGGcAGccAAAcCuCAuUdTsdT 35 A-108815.3 AAUGAGGUuUGGCUGcCugdTsdT 38108813.2 TFR2 AD-52589 239 A-99598.5 cAGGcAGccAAAccucAuudTsdT 35A-108810.1 AAUGAGGUuUGGCuGcCuGdTsdT 38 TFR2 AD-52590 239 A-cAGGcAGcCAAAcCuCAuUdTsdT 35 A-108815.4 AAUGAGGUuUGGCUGcCugdTsdT 38108814.2 It should be noted that unmodified versions of each of themodified sequences shown are included within the scope of the invention.

TABLE 14 TFR2 Dose Response Table 14 Target Reactivity Duplex Name StartPosition IC50 (nM) TFR2 Human AD-47814 64 0.019 TFR2 Human AD-52549 640.034 TFR2 Human AD-52572 64 0.059 TFR2 Human AD-52550 64 0.062 TFR2Human AD-52573 64 0.102 TFR2 Human AD-52570 239 0.035 TFR2 HumanAD-47826 239 0.036 TFR2 Human AD-52590 239 0.038 TFR2 Human AD-52574 2390.065 TFR2 Human AD-52558 239 0.236

TABLE 15 SMAD4 Unmodified Duplexes Table 15 SEQ SEQ Duplex Sense OligoID Antis Oligo ID Name Start Target Name Trans Seq NO: Name Trans SeqNO: AD-48090.1 481 SMAD4 A-100350.1 AUGCCUGUCUGAGCAUUGU 884 A-100351.1ACAAUGCUCAGACAGGCAU 929 AD-48091.1 772 SMAD4 A-100366.1AUGUUAAAUAUUGUCAGUA 885 A-100367.1 UACUGACAAUAUUUAACAU 930 AD-48092.1817 SMAD4 A-100382.1 UCUGUGUGAAUCCAUAUCA 886 A-100383.1UGAUAUGGAUUCACACAGA 931 AD-48093.1 1212 SMAD4 A-100398.1ACUUACCAUCAUAACAGCA 887 A-100399.1 UGCUGUUAUGAUGGUAAGU 932 AD-48094.11351 SMAD4 A-100414.1 ACAAUGAGCUUGCAUUCCA 888 A-100415.1UGGAAUGCAAGCUCAUUGU 933 AD-48095.1 1712 SMAD4 A-100430.1UGUUCAUAAGAUCUACCCA 889 A-100431.1 UGGGUAGAUCUUAUGAACA 934 AD-48096.1590 SMAD4 A-100352.1 AAAAGAUGAAUUGGAUUCU 890 A-100353.1AGAAUCCAAUUCAUCUUUU 935 AD-48097.1 773 SMAD4 A-100368.1UGUUAAAUAUUGUCAGUAU 891 A-100369.1 AUACUGACAAUAUUUAACA 936 AD-48098.1819 SMAD4 A-100384.1 UGUGUGAAUCCAUAUCACU 892 A-100385.1AGUGAUAUGGAUUCACACA 937 AD-48099.1 1232 SMAD4 A-100400.1UACCACCUGGACUGGAAGU 893 A-100401.1 ACUUCCAGUCCAGGUGGUA 938 AD-48100.11362 SMAD4 A-100416.1 GCAUUCCAGCCUCCCAUUU 894 A-100417.1AAAUGGGAGGCUGGAAUGC 939 AD-48101.1 1713 SMAD4 A-100432.1GUUCAUAAGAUCUACCCAA 895 A-100433.1 UUGGGUAGAUCUUAUGAAC 940 AD-48102.1602 SMAD4 A-100354.1 GGAUUCUUUAAUAACAGCU 896 A-100355.1AGCUGUUAUUAAAGAAUCC 941 AD-48103.1 777 SMAD4 A-100370.1AAAUAUUGUCAGUAUGCGU 897 A-100371.1 ACGCAUACUGACAAUAUUU 942 AD-48104.1820 SMAD4 A-100386.1 GUGUGAAUCCAUAUCACUA 898 A-100387.1UAGUGAUAUGGAUUCACAC 943 AD-48105.1 1238 SMAD4 A-100402.1CUGGACUGGAAGUAGGACU 899 A-100403.1 AGUCCUACUUCCAGUCCAG 944 AD-48106.11367 SMAD4 A-100418.1 CCAGCCUCCCAUUUCCAAU 900 A-100419.1AUUGGAAAUGGGAGGCUGG 945 AD-48107.1 2816 SMAD4 A-100434.1UAUUUCUAGGCACAAGGUU 901 A-100435.1 AACCUUGUGCCUAGAAAUA 946 AD-48108.1608 SMAD4 A-100356.1 UUUAAUAACAGCUAUAACU 902 A-100357.1AGUUAUAGCUGUUAUUAAA 947 AD-48109.1 778 SMAD4 A-100372.1AAUAUUGUCAGUAUGCGUU 903 A-100373.1 AACGCAUACUGACAAUAUU 948 AD-48110.1861 SMAD4 A-100388.1 AUUGAUCUCUCAGGAUUAA 904 A-100389.1UUAAUCCUGAGAGAUCAAU 949 AD-48111.1 1250 SMAD4 A-100404.1UAGGACUGCACCAUACACA 905 A-100405.1 UGUGUAUGGUGCAGUCCUA 950 AD-48112.11370 SMAD4 A-100420.1 GCCUCCCAUUUCCAAUCAU 906 A-100421.1AUGAUUGGAAAUGGGAGGC 951 AD-48113.1 2984 SMAD4 A-100436.1AAUAUUUUGGAAACUGCUA 907 A-100437.1 UAGCAGUUUCCAAAAUAUU 952 AD-48114.1611 SMAD4 A-100358.1 AAUAACAGCUAUAACUACA 908 A-100359.1UGUAGUUAUAGCUGUUAUU 953 AD-48115.1 781 SMAD4 A-100374.1AUUGUCAGUAUGCGUUUGA 909 A-100375.1 UCAAACGCAUACUGACAAU 954 AD-48116.11090 SMAD4 A-100390.1 CUGUGGCUUCCACAAGUCA 910 A-100391.1UGACUUGUGGAAGCCACAG 955 AD-48117.1 1257 SMAD4 A-100406.1GCACCAUACACACCUAAUU 911 A-100407.1 AAUUAGGUGUGUAUGGUGC 956 AD-48118.11601 SMAD4 A-100422.1 GUUGGAAUGUAAAGGUGAA 912 A-100423.1UUCACCUUUACAUUCCAAC 957 AD-48119.1 3013 SMAD4 A-100438.1UAAAUACUGUGCAGAAUAA 913 A-100439.1 UUAUUCUGCACAGUAUUUA 958 AD-48120.1659 SMAD4 A-100360.1 CAUACAGAGAACAUUGGAU 914 A-100361.1AUCCAAUGUUCUCUGUAUG 959 AD-48121.1 783 SMAD4 A-100376.1UGUCAGUAUGCGUUUGACU 915 A-100377.1 AGUCAAACGCAUACUGACA 960 AD-48122.11137 SMAD4 A-100392.1 AGUGAAGGACUGUUGCAGA 916 A-100393.1UCUGCAACAGUCCUUCACU 961 AD-48123.1 1262 SMAD4 A-100408.1AUACACACCUAAUUUGCCU 917 A-100409.1 AGGCAAAUUAGGUGUGUAU 962 AD-48124.11633 SMAD4 A-100424.1 UCAGGUGCCUUAGUGACCA 918 A-100425.1UGGUCACUAAGGCACCUGA 963 AD-48125.1 698 SMAD4 A-100362.1UCGGAAAGGAUUUCCUCAU 919 A-100363.1 AUGAGGAAAUCCUUUCCGA 964 AD-48126.1784 SMAD4 A-100378.1 GUCAGUAUGCGUUUGACUU 920 A-100379.1AAGUCAAACGCAUACUGAC 965 AD-48126.2 784 SMAD4 A-100378.2GUCAGUAUGCGUUUGACUU 920 A-100379.2 AAGUCAAACGCAUACUGAC 965 AD-48127.11207 SMAD4 A-100394.1 CAGCUACUUACCAUCAUAA 921 A-100395.1UUAUGAUGGUAAGUAGCUG 966 AD-48128.1 1272 SMAD4 A-100410.1AAUUUGCCUCACCACCAAA 922 A-100411.1 UUUGGUGGUGAGGCAAAUU 967 AD-48129.11650 SMAD4 A-100426.1 CACGCGGUCUUUGUACAGA 923 A-100427.1UCUGUACAAAGACCGCGUG 968 AD-48130.1 771 SMAD4 A-100364.1CAUGUUAAAUAUUGUCAGU 924 A-100365.1 ACUGACAAUAUUUAACAUG 969 AD-48131.1791 SMAD4 A-100380.1 UGCGUUUGACUUAAAAUGU 925 A-100381.1ACAUUUUAAGUCAAACGCA 970 AD-48132.1 1209 SMAD4 A-100396.1GCUACUUACCAUCAUAACA 926 A-100397.1 UGUUAUGAUGGUAAGUAGC 971 AD-48133.11273 SMAD4 A-100412.1 AUUUGCCUCACCACCAAAA 927 A-100413.1UUUUGGUGGUGAGGCAAAU 972 AD-48134.1 1652 SMAD4 A-100428.1CGCGGUCUUUGUACAGAGU 928 A-100429.1 ACUCUGUACAAAGACCGCG 973 Note that anoverhang (e.g. TT, dTsdT) can be added to the 3′ end of any duplex.

TABLE 16 SMAD4 Modified Duplexes Table Sense SEQ SEQ 16 Duplex Oligo IDAntis Oligo ID Target Name Start Name Oligo Seq NO: Name Oligo Seq NO:SMAD4 AD-48090.1 481 A-100350.1 AuGccuGucuGAGcAuuGud 974 A-100351.1AcAAUGCUcAGAcAGGcAUdTsdT 1020 TsdT SMAD4 AD-48091.1 772 A-100366.1AuGuuAAAuAuuGucAGuAd 975 A-100367.1 uACUGAcAAuAUUuAAcAUdTsdT 1021 TsdTSMAD4 AD-48092.1 817 A-100382.1 ucuGuGuGAAuccAuAucAd 976 A-100383.1UGAuAUGGAUUcAcAcAGAdTsdT 1022 TsdT SMAD4 AD-48093.1 1212 A-100398.1AcuuAccAucAuAAcAGcAd 977 A-100399.1 UGCUGUuAUGAUGGuAAGUdTsdT 1023 TsdTSMAD4 AD-48094.1 1351 A-100414.1 AcAAuGAGcuuGcAuuccAd 978 A-100415.1UGGAAUGcAAGCUcAUUGUdTsdT 1024 TsdT SMAD4 AD-48095.1 1712 A-100430.1uGuucAuAAGAucuAcccAd 979 A-100431.1 UGGGuAGAUCUuAUGAAcAdTsdT 1025 TsdTSMAD4 AD-48096.1 590 A-100352.1 AAAAGAuGAAuuGGAuucud 980 A-100353.1AGAAUCcAAUUcAUCUUUUdTsdT 1026 TsdT SMAD4 AD-48097.1 773 A-100368.1uGuuAAAuAuuGucAGuAud 981 A-100369.1 AuACUGAcAAuAUUuAAcAdTsdT 1027 TsdTSMAD4 AD-48098.1 819 A-100384.1 uGuGuGAAuccAuAucAcud 982 A-100385.1AGUGAuAUGGAUUcAcAcAdTsdT 1028 TsdT SMAD4 AD-48099.1 1232 A-100400.1uAccAccuGGAcuGGAAGud 983 A-100401.1 ACUUCcAGUCcAGGUGGuAdTsdT 1029 TsdTSMAD4 AD-48100.1 1362 A-100416.1 GcAuuccAGccucccAuuud 984 A-100417.1AAAUGGGAGGCUGGAAUGCdTsdT 1030 TsdT SMAD4 AD-48101.1 1713 A-100432.1GuucAuAAGAucuAcccAAd 985 A-100433.1 UUGGGuAGAUCUuAUGAACdTsdT 1031 TsdTSMAD4 AD-48102.1 602 A-100354.1 GGAuucuuuAAuAAcAGcud 986 A-100355.1AGCUGUuAUuAAAGAAUCCdTsdT 1032 TsdT SMAD4 AD-48103.1 777 A-100370.1AAAuAuuGucAGuAuGcGud 987 A-100371.1 ACGcAuACUGAcAAuAUUUdTsdT 1033 TsdTSMAD4 AD-48104.1 820 A-100386.1 GuGuGAAuccAuAucAcuAd 988 A-100387.1uAGUGAuAUGGAUUcAcACdTsdT 1034 TsdT SMAD4 AD-48105.1 1238 A-100402.1cuGGAcuGGAAGuAGGAcud 989 A-100403.1 AGUCCuACUUCcAGUCcAGdTsdT 1035 TsdTSMAD4 AD-48106.1 1367 A-100418.1 ccAGccucccAuuuccAAud 990 A-100419.1AUUGGAAAUGGGAGGCUGGdTsdT 1036 TsdT SMAD4 AD-48107.1 2816 A-100434.1uAuuucuAGGcAcAAGGuud 991 A-100435.1 AACCUUGUGCCuAGAAAuAdTsdT 1037 TsdTSMAD4 AD-48108.1 608 A-100356.1 uuuAAuAAcAGcuAuAAcud 992 A-100357.1AGUuAuAGCUGUuAUuAAAdTsdT 1038 TsdT SMAD4 AD-48109.1 778 A-100372.1AAuAuuGucAGuAuGcGuud 993 A-100373.1 AACGcAuACUGAcAAuAUUdTsdT 1039 TsdTSMAD4 AD-48110.1 861 A-100388.1 AuuGAucucucAGGAuuAAd 994 A-100389.1UuAAUCCUGAGAGAUcAAUdTsdT 1040 TsdT SMAD4 AD-48111.1 1250 A-100404.1uAGGAcuGcAccAuAcAcAd 995 A-100405.1 UGUGuAUGGUGcAGUCCuAdTsdT 1041 TsdTSMAD4 AD-48112.1 1370 A-100420.1 GccucccAuuuccAAucAud 996 A-100421.1AUGAUUGGAAAUGGGAGGCdTsdT 1042 TsdT SMAD4 AD-48113.1 2984 A-100436.1AAuAuuuuGGAAAcuGcuAd 997 A-100437.1 uAGcAGUUUCcAAAAuAUUdTsdT 1043 TsdTSMAD4 AD-48114.1 611 A-100358.1 AAuAAcAGcuAuAAcuAcAd 998 A-100359.1UGuAGUuAuAGCUGUuAUUdTsdT 1044 TsdT SMAD4 AD-48115.1 781 A-100374.1AuuGucAGuAuGcGuuuGAd 999 A-100375.1 UcAAACGcAuACUGAcAAUdTsdT 1045 TsdTSMAD4 AD-48116.1 1090 A-100390.1 cuGuGGcuuccAcAAGucAd 1000 A-100391.1UGACUUGUGGAAGCcAcAGdTsdT 1046 TsdT SMAD4 AD-48117.1 1257 A-100406.1GcAccAuAcAcAccuAAuud 1001 A-100407.1 AAUuAGGUGUGuAUGGUGCdTsdT 1047 TsdTSMAD4 AD-48118.1 1601 A-100422.1 GuuGGAAuGuAAAGGuGAAd 1002 A-100423.1UUcACCUUuAcAUUCcAACdTsdT 1048 TsdT SMAD4 AD-48119.1 3013 A-100438.1uAAAuAcuGuGcAGAAuAAd 1003 A-100439.1 UuAUUCUGcAcAGuAUUuAdTsdT 1049 TsdTSMAD4 AD-48120.1 659 A-100360.1 cAuAcAGAGAAcAuuGGAud 1004 A-100361.1AUCcAAUGUUCUCUGuAUGdTsdT 1050 TsdT SMAD4 AD-48121.1 783 A-100376.1uGucAGuAuGcGuuuGAcud 1005 A-100377.1 AGUcAAACGcAuACUGAcAdTsdT 1051 TsdTSMAD4 AD-48122.1 1137 A-100392.1 AGuGAAGGAcuGuuGcAGAd 1006 A-100393.1UCUGcAAcAGUCCUUcACUdTsdT 1052 TsdT SMAD4 AD-48123.1 1262 A-100408.1AuAcAcAccuAAuuuGccud 1007 A-100409.1 AGGcAAAUuAGGUGUGuAUdTsdT 1053 TsdTSMAD4 AD-48124.1 1633 A-100424.1 ucAGGuGccuuAGuGAccAd 1008 A-100425.1UGGUcACuAAGGcACCUGAdTsdT 1054 TsdT SMAD4 AD-48125.1 698 A-100362.1ucGGAAAGGAuuuccucAud 1009 A-100363.1 AUGAGGAAAUCCUUUCCGAdTsdT 1055 TsdTSMAD4 AD-48126.1 784 A-100378.1 GucAGuAuGcGuuuGAcuud 1010 A-100379.1AAGUcAAACGcAuACUGACdTsdT 1056 TsdT SMAD4 AD-48126.2 784 A-100378.2GucAGuAuGcGuuuGAcuud 1011 A-100379.2 AAGUcAAACGcAuACUGACdTsdT 1057 TsdTSMAD4 AD-48127.1 1207 A-100394.1 cAGcuAcuuAccAucAuAAd 1012 A-100395.1UuAUGAUGGuAAGuAGCUGdTsdT 1058 TsdT SMAD4 AD-48128.1 1272 A-100410.1AAuuuGccucAccAccAAAd 1013 A-100411.1 UUUGGUGGUGAGGcAAAUUdTsdT 1059 TsdTSMAD4 AD-48129.1 1650 A-100426.1 cAcGcGGucuuuGuAcAGAd 1014 A-100427.1UCUGuAcAAAGACCGCGUGdTsdT 1060 TsdT SMAD4 AD-48130.1 771 A-100364.1cAuGuuAAAuAuuGucAGud 1015 A-100365.1 ACUGAcAAuAUUuAAcAUGdTsdT 1061 TsdTSMAD4 AD-48131.1 791 A-100380.1 uGcGuuuGAcuuAAAAuGud 1016 A-100381.1AcAUUUuAAGUcAAACGcAdTsdT 1062 TsdT SMAD4 AD-48132.1 1209 A-100396.1GcuAcuuAccAucAuAAcAd 1017 A-100397.1 UGUuAUGAUGGuAAGuAGCdTsdT 1063 TsdTSMAD4 AD-48133.1 1273 A-100412.1 AuuuGccucAccAccAAAAd 1018 A-100413.1UUUUGGUGGUGAGGcAAAUdTsdT 1064 TsdT SMAD4 AD-48134.1 1652 A-100428.1cGcGGucuuuGuAcAGAGud 1019 A-100429.1 ACUCUGuAcAAAGACCGCGdTsdT 1065 TsdTIt should be noted that unmodified versions of each of the modifiedsequences shown are included within the scope of the invention.

TABLE 17 NEO1 Unmodified Duplexes Table 17 Duplex Sense SEQ ID AntisOligo SEQ ID Target Name Start OligoName Trans Seq NO: Name Trans SeqNO: NEO1 AD-48273.1 4618 A-100622.1 CUCCGAGAGUAGCUAUGAA 1066 A-100623.1UUCAUAGCUACUCUCGGAG 1110 NEO1 AD-48287.1 546 A-100564.1GCUCUUCUGUUAUAUUAAA 1067 A-100565.1 UUUAAUAUAACAGAAGAGC 1111 NEO1AD-48274.1 5060 A-100638.1 GAGUGUAGACAUUGGCAUU 1068 A-100639.1AAUGCCAAUGUCUACACUC 1112 NEO1 AD-48309.1 4778 A-100634.1GGAAUUGUACAGAGUACGA 1069 A-100635.1 UCGUACUCUGUACAAUUCC 1113 NEO1AD-48309.2 4778 A-100634.2 GGAAUUGUACAGAGUACGA 1070 A-100635.2UCGUACUCUGUACAAUUCC 1114 NEO1 AD-48297.1 4674 A-100630.1GACUAAUGAAGGACCUAAA 1071 A-100631.1 UUUAGGUCCUUCAUUAGUC 1115 NEO1AD-48296.1 4495 A-100614.1 GAACCAUCACAUUCACUCA 1072 A-100615.1UGAGUGAAUGUGAUGGUUC 1116 NEO1 AD-48280.1 5062 A-100640.1GUGUAGACAUUGGCAUUUA 1073 A-100641.1 UAAAUGCCAAUGUCUACAC 1117 NEO1AD-48275.1 535 A-100560.1 CUCAGUUAGAGGCUCUUCU 1074 A-100561.1AGAAGAGCCUCUAACUGAG 1118 NEO1 AD-48276.1 1283 A-100576.1GAUGAUGCUGGGACUUAUU 1075 A-100577.1 AAUAAGUCCCAGCAUCAUC 1119 NEO1AD-48269.1 533 A-100558.1 CUCUCAGUUAGAGGCUCUU 1076 A-100559.1AAGAGCCUCUAACUGAGAG 1120 NEO1 AD-48286.1 5069 A-100642.1CAUUGGCAUUUAUGUACAA 1077 A-100643.1 UUGUACAUAAAUGCCAAUG 1121 NEO1AD-48299.1 791 A-100568.1 GCAGGUCUUCCAAGAUUUA 1078 A-100569.1UAAAUCUUGGAAGACCUGC 1122 NEO1 AD-48295.1 2602 A-100598.1CCUAGAUGAAACUCGUGUU 1079 A-100599.1 AACACGAGUUUCAUCUAGG 1123 NEO1AD-48292.1 5329 A-100644.1 GCAUUGCUGUUUGUAAGCU 1080 A-100645.1AGCUUACAAACAGCAAUGC 1124 NEO1 AD-48293.1 686 A-100566.1GUGGUGCAUUCCAAACACA 1081 A-100567.1 UGUGUUUGGAAUGCACCAC 1125 NEO1AD-48288.1 1535 A-100580.1 GUUUUGGGUCUGGUGAAAU 1082 A-100581.1AUUUCACCAGACCCAAAAC 1126 NEO1 AD-48307.1 4066 A-100602.1GCCUGUGAUUAGUGCCCAU 1083 A-100603.1 AUGGGCACUAAUCACAGGC 1127 NEO1AD-48270.1 1282 A-100574.1 GGAUGAUGCUGGGACUUAU 1084 A-100575.1AUAAGUCCCAGCAUCAUCC 1128 NEO1 AD-48300.1 1949 A-100584.1GCUCAAAAUAAGCAUGGCU 1085 A-100585.1 AGCCAUGCUUAUUUUGAGC 1129 NEO1AD-48306.1 2227 A-100586.1 CCGAGUGGUGGCCUACAAU 1086 A-100587.1AUUGUAGGCCACCACUCGG 1130 NEO1 AD-48315.1 5059 A-100636.1GGAGUGUAGACAUUGGCAU 1087 A-100637.1 AUGCCAAUGUCUACACUCC 1131 NEO1AD-48291.1 4673 A-100628.1 GGACUAAUGAAGGACCUAA 1088 A-100629.1UUAGGUCCUUCAUUAGUCC 1132 NEO1 AD-48272.1 4096 A-100606.1CCUCGAUAACCCUCACCAU 1089 A-100607.1 AUGGUGAGGGUUAUCGAGG 1133 NEO1AD-48271.1 2273 A-100590.1 GAUGUUGCUGUUCGAACAU 1090 A-100591.1AUGUUCGAACAGCAACAUC 1134 NEO1 AD-48294.1 1540 A-100582.1GGGUCUGGUGAAAUCAGAU 1091 A-100583.1 AUCUGAUUUCACCAGACCC 1135 NEO1AD-48278.1 4123 A-100608.1 CUCCAGCAGCCUCGCUUCU 1092 A-100609.1AGAAGCGAGGCUGCUGGAG 1136 NEO1 AD-48277.1 2312 A-100592.1GCUCCUCAGAAUCUGUCCU 1093 A-100593.1 AGGACAGAUUCUGAGGAGC 1137 NEO1AD-48313.1 4086 A-100604.1 CCAUCCAUUCCCUCGAUAA 1094 A-100605.1UUAUCGAGGGAAUGGAUGG 1138 NEO1 AD-48289.1 2484 A-100596.1CUCAGCUGAUUGAAGGUCU 1095 A-100597.1 AGACCUUCAAUCAGCUGAG 1139 NEO1AD-48290.1 4179 A-100612.1 GGCCCAUUGGCACAUCCAU 1096 A-100613.1AUGGAUGUGCCAAUGGGCC 1140 NEO1 AD-48284.1 4174 A-100610.1CCCAUGGCCCAUUGGCACA 1097 A-100611.1 UGUGCCAAUGGGCCAUGGG 1141 NEO1AD-48298.1 6731 A-100646.1 GUACCUGGAUACUGCCACA 1098 A-100647.1UGUGGCAGUAUCCAGGUAC 1142 NEO1 AD-48311.1 852 A-100572.1CAAUUCUGAAUUGUGAAGU 1099 A-100573.1 ACUUCACAAUUCAGAAUUG 1143 NEO1AD-48285.1 4664 A-100626.1 CACCUGGAAGGACUAAUGA 1100 A-100627.1UCAUUAGUCCUUCCAGGUG 1144 NEO1 AD-48282.1 1448 A-100578.1CCAACUCCAACUGUGAAGU 1101 A-100579.1 ACUUCACAGUUGGAGUUGG 1145 NEO1AD-48302.1 4542 A-100616.1 GAAGGAGCCGGCCUCCUAU 1102 A-100617.1AUAGGAGGCCGGCUCCUUC 1146 NEO1 AD-48303.1 4767 A-100632.1CUUGAAAACAAGGAAUUGU 1103 A-100633.1 ACAAUUCCUUGUUUUCAAG 1147 NEO1AD-48279.1 4629 A-100624.1 GCUAUGAACCAGAUGAGCU 1104 A-100625.1AGCUCAUCUGGUUCAUAGC 1148 NEO1 AD-48301.1 3361 A-100600.1GAUACAUGACUGGGUUAUU 1105 A-100601.1 AAUAACCCAGUCAUGUAUC 1149 NEO1AD-48314.1 4613 A-100620.1 GAAGACUCCGAGAGUAGCU 1106 A-100621.1AGCUACUCUCGGAGUCUUC 1150 NEO1 AD-48312.1 2236 A-100588.1GGCCUACAAUAAACAUGGU 1107 A-100589.1 ACCAUGUUUAUUGUAGGCC 1151 NEO1AD-48304.1 7033 A-100648.1 GUACACACUUGUUUGGCCU 1108 A-100649.1AGGCCAAACAAGUGUGUAC 1152 NEO1 AD-48310.1 7043 A-100650.1GUUUGGCCUUUUCUGUAGU 1109 A-100651.1 ACUACAGAAAAGGCCAAAC 1153 Note thatan overhang (e.g. TT, dTsdT) can be added to the 3′ end of any duplex.

TABLE 18 NEO1 Modified Duplexes Table 18 SEQ SEQ Duplex Sense Oligo IDAntis Oligo ID Name Target Start Name Oligo Seq NO: Name Oligo Seq NO:AD-48273.1 NEO1 4618 A-100622.1 cuccGAGAGuAGcuAuGAAd 1154 A-100623.1UUcAuAGCuACUCUCGGAGdTsdT 1198 TsdT AD-48287.1 NEO1 546 A-100564.1GcucuucuGuuAuAuuAAAd 1155 A-100565.1 UUuAAuAuAAcAGAAGAGCdTsdT 1199 TsdTAD-48274.1 NEO1 5060 A-100638.1 GAGuGuAGAcAuuGGcAuud 1156 A-100639.1AAUGCcAAUGUCuAcACUCdTsdT 1200 TsdT AD-48309.1 NEO1 4778 A-100634.1GGAAuuGuAcAGAGuAcGAd 1157 A-100635.1 UCGuACUCUGuAcAAUUCCdTsdT 1201 TsdTAD-48309.2 NEO1 4778 A-100634.2 GGAAuuGuAcAGAGuAcGAd 1158 A-100635.2UCGuACUCUGuAcAAUUCCdTsdT 1202 TsdT AD-48297.1 NEO1 4674 A-100630.1GAcuAAuGAAGGAccuAAAd 1159 A-100631.1 UUuAGGUCCUUcAUuAGUCdTsdT 1203 TsdTAD-48296.1 NEO1 4495 A-100614.1 GAAccAucAcAuucAcucAd 1160 A-100615.1UGAGUGAAUGUGAUGGUUCdTsdT 1204 TsdT AD-48280.1 NEO1 5062 A-100640.1GuGuAGAcAuuGGcAuuuAd 1161 A-100641.1 uAAAUGCcAAUGUCuAcACdTsdT 1205 TsdTAD-48275.1 NEO1 535 A-100560.1 cucAGuuAGAGGcucuucud 1162 A-100561.1AGAAGAGCCUCuAACUGAGdTsdT 1206 TsdT AD-48276.1 NEO1 1283 A-100576.1GAuGAuGcuGGGAcuuAuud 1163 A-100577.1 AAuAAGUCCcAGcAUcAUCdTsdT 1207 TsdTAD-48269.1 NEO1 533 A-100558.1 cucucAGuuAGAGGcucuud 1164 A-100559.1AAGAGCCUCuAACUGAGAGdTsdT 1208 TsdT AD-48286.1 NEO1 5069 A-100642.1cAuuGGcAuuuAuGuAcAAd 1165 A-100643.1 UUGuAcAuAAAUGCcAAUGdTsdT 1209 TsdTAD-48299.1 NEO1 791 A-100568.1 GcAGGucuuccAAGAuuuAd 1166 A-100569.1uAAAUCUUGGAAGACCUGCdTsdT 1210 TsdT AD-48295.1 NEO1 2602 A-100598.1ccuAGAuGAAAcucGuGuud 1167 A-100599.1 AAcACGAGUUUcAUCuAGGdTsdT 1211 TsdTAD-48292.1 NEO1 5329 A-100644.1 GcAuuGcuGuuuGuAAGcud 1168 A-100645.1AGCUuAcAAAcAGcAAUGCdTsdT 1212 TsdT AD-48293.1 NEO1 686 A-100566.1GuGGuGcAuuccAAAcAcAd 1169 A-100567.1 UGUGUUUGGAAUGcACcACdTsdT 1213 TsdTAD-48288.1 NEO1 1535 A-100580.1 GuuuuGGGucuGGuGAAAud 1170 A-100581.1AUUUcACcAGACCcAAAACdTsdT 1214 TsdT AD-48307.1 NEO1 4066 A-100602.1GccuGuGAuuAGuGcccAud 1171 A-100603.1 AUGGGcACuAAUcAcAGGCdTsdT 1215 TsdTAD-48270.1 NEO1 1282 A-100574.1 GGAuGAuGcuGGGAcuuAud 1172 A-100575.1AuAAGUCCcAGcAUcAUCCdTsdT 1216 TsdT AD-48300.1 NEO1 1949 A-100584.1GcucAAAAuAAGcAuGGcud 1173 A-100585.1 AGCcAUGCUuAUUUUGAGCdTsdT 1217 TsdTAD-48306.1 NEO1 2227 A-100586.1 ccGAGuGGuGGccuAcAAud 1174 A-100587.1AUUGuAGGCcACcACUCGGdTsdT 1218 TsdT AD-48315.1 NEO1 5059 A-100636.1GGAGuGuAGAcAuuGGcAud 1175 A-100637.1 AUGCcAAUGUCuAcACUCCdTsdT 1219 TsdTAD-48291.1 NEO1 4673 A-100628.1 GGAcuAAuGAAGGAccuAAd 1176 A-100629.1UuAGGUCCUUcAUuAGUCCdTsdT 1220 TsdT AD-48272.1 NEO1 4096 A-100606.1ccucGAuAAcccucAccAud 1177 A-100607.1 AUGGUGAGGGUuAUCGAGGdTsdT 1221 TsdTAD-48271.1 NEO1 2273 A-100590.1 GAuGuuGcuGuucGAAcAud 1178 A-100591.1AUGUUCGAAcAGcAAcAUCdTsdT 1222 TsdT AD-48294.1 NEO1 1540 A-100582.1GGGucuGGuGAAAucAGAud 1179 A-100583.1 AUCUGAUUUcACcAGACCCdTsdT 1223 TsdTAD-48278.1 NEO1 4123 A-100608.1 cuccAGcAGccucGcuucud 1180 A-100609.1AGAAGCGAGGCUGCUGGAGdTsdT 1224 TsdT AD-48277.1 NEO1 2312 A-100592.1GcuccucAGAAucuGuccud 1181 A-100593.1 AGGAcAGAUUCUGAGGAGCdTsdT 1225 TsdTAD-48313.1 NEO1 4086 A-100604.1 ccAuccAuucccucGAuAAd 1182 A-100605.1UuAUCGAGGGAAUGGAUGGdTsdT 1226 TsdT AD-48289.1 NEO1 2484 A-100596.1cucAGcuGAuuGAAGGucud 1183 A-100597.1 AGACCUUcAAUcAGCUGAGdTsdT 1227 TsdTAD-48290.1 NEO1 4179 A-100612.1 GGcccAuuGGcAcAuccAud 1184 A-100613.1AUGGAUGUGCcAAUGGGCCdTsdT 1228 TsdT AD-48284.1 NEO1 4174 A-100610.1cccAuGGcccAuuGGcAcAd 1185 A-100611.1 UGUGCcAAUGGGCcAUGGGdTsdT 1229 TsdTAD-48298.1 NEO1 6731 A-100646.1 GuAccuGGAuAcuGccAcAd 1186 A-100647.1UGUGGcAGuAUCcAGGuACdTsdT 1230 TsdT AD-48311.1 NEO1 852 A-100572.1cAAuucuGAAuuGuGAAGud 1187 A-100573.1 ACUUcAcAAUUcAGAAUUGdTsdT 1231 TsdTAD-48285.1 NEO1 4664 A-100626.1 cAccuGGAAGGAcuAAuGAd 1188 A-100627.1UcAUuAGUCCUUCcAGGUGdTsdT 1232 TsdT AD-48282.1 NEO1 1448 A-100578.1ccAAcuccAAcuGuGAAGud 1189 A-100579.1 ACUUcAcAGUUGGAGUUGGdTsdT 1233 TsdTAD-48302.1 NEO1 4542 A-100616.1 GAAGGAGccGGccuccuAud 1190 A-100617.1AuAGGAGGCCGGCUCCUUCdTsdT 1234 TsdT AD-48303.1 NEO1 4767 A-100632.1cuuGAAAAcAAGGAAuuGud 1191 A-100633.1 AcAAUUCCUUGUUUUcAAGdTsdT 1235 TsdTAD-48279.1 NEO1 4629 A-100624.1 GcuAuGAAccAGAuGAGcud 1192 A-100625.1AGCUcAUCUGGUUcAuAGCdTsdT 1236 TsdT AD-48301.1 NEO1 3361 A-100600.1GAuAcAuGAcuGGGuuAuud 1193 A-100601.1 AAuAACCcAGUcAUGuAUCdTsdT 1237 TsdTAD-48314.1 NEO1 4613 A-100620.1 GAAGAcuccGAGAGuAGcud 1194 A-100621.1AGCuACUCUCGGAGUCUUCdTsdT 1238 TsdT AD-48312.1 NEO1 2236 A-100588.1GGccuAcAAuAAAcAuGGud 1195 A-100589.1 ACcAUGUUuAUUGuAGGCCdTsdT 1239 TsdTAD-48304.1 NEO1 7033 A-100648.1 GuAcAcAcuuGuuuGGccud 1196 A-100649.1AGGCcAAAcAAGUGUGuACdTsdT 1240 TsdT AD-48310.1 NEO1 7043 A-100650.1GuuuGGccuuuucuGuAGud 1197 A-100651.1 ACuAcAGAAAAGGCcAAACdTsdT 1241 TsdTIt should be noted that unmodified versions of each of the modifiedsequences shown are included within the scope of the invention.

TABLE 19 SMAD4 Percent Inhibition Table 19 0.1 nM (% message 10 nM (%message remaining) remaining) Target ID Avg SD Avg SD SMAD4 AD-4809093.6 4.6 54.6 5.6 SMAD4 AD-48091 98.0 5.0 60.8 3.3 SMAD4 AD-48092 64.60.2 47.8 12.0 SMAD4 AD-48093 96.4 3.5 45.0 8.0 SMAD4 AD-48094 41.3 0.416.3 5.4 SMAD4 AD-48095 64.4 9.1 30.0 0.5 SMAD4 AD-48096 70.5 1.8 44.30.7 SMAD4 AD-48097 19.6 2.5 10.0 1.6 SMAD4 AD-48098 60.6 2.1 29.9 1.8SMAD4 AD-48099 83.1 5.5 57.2 2.5 SMAD4 AD-48100 73.4 1.6 50.4 1.2 SMAD4AD-48101 34.8 3.7 23.3 0.9 SMAD4 AD-48102 66.9 3.2 35.5 4.0 SMAD4AD-48103 43.4 8.9 20.5 1.0 SMAD4 AD-48104 53.5 6.2 20.5 1.5 SMAD4AD-48105 59.4 0.6 23.8 3.0 SMAD4 AD-48106 68.4 0.3 40.7 0.5 SMAD4AD-48107 40.9 3.0 26.9 6.6 SMAD4 AD-48108 21.4 4.3 15.2 4.3 SMAD4AD-48109 19.2 4.1 12.1 5.2 SMAD4 AD-48110 46.1 6.4 28.4 8.1 SMAD4AD-48111 75.9 5.1 68.4 12.1 SMAD4 AD-48112 75.8 2.0 72.0 10.4 SMAD4AD-48113 87.4 11.1 72.0 2.7 SMAD4 AD-48114 36.7 3.2 19.2 0.6 SMAD4AD-48115 35.8 2.8 18.6 1.9 SMAD4 AD-48116 37.1 0.2 13.6 0.9 SMAD4AD-48117 32.1 0.8 21.1 1.4 SMAD4 AD-48118 26.3 1.1 16.4 5.5 SMAD4AD-48119 52.1 4.7 38.8 4.5 SMAD4 AD-48120 32.1 1.0 13.9 1.4 SMAD4AD-48121 24.3 2.3 10.0 0.7 SMAD4 AD-48122 31.4 5.7 14.6 1.7 SMAD4AD-48123 27.4 1.5 14.6 2.2 SMAD4 AD-48124 76.8 7.0 55.8 1.0 SMAD4AD-48125 28.7 2.6 12.6 0.9 SMAD4 AD-48126 18.9 1.9 7.4 0.2 SMAD4AD-48127 67.5 3.7 39.6 4.0 SMAD4 AD-48128 69.8 4.0 44.5 6.1 SMAD4AD-48129 73.1 3.4 42.6 2.0 SMAD4 AD-48130 18.1 0.1 12.5 0.9 SMAD4AD-48131 44.4 0.5 17.1 4.1 SMAD4 AD-48132 47.7 0.1 22.6 5.4 SMAD4AD-48133 57.1 1.8 30.4 10.0 SMAD4 AD-48134 86.3 18.0 42.4 9.2

TABLE 20 NEO1 Percent Inhibition Table 20 0.1 nM (% message 10 nM (%message remaining) remaining) Target ID Avg SD Avg SD Neo1 AD-48273 8.40.7 9.3 3.6 Neo1 AD-48287 8.6 5.5 10.4 2.7 Neo1 AD-48274 11.0 4.3 6.52.2 Neo1 AD-48309 11.0 0.6 6.5 0.8 Neo1 AD-48297 12.9 1.6 8.7 2.4 Neo1AD-48296 14.0 6.9 7.6 0.1 Neo1 AD-48280 15.6 3.7 10.8 7.1 Neo1 AD-4827517.7 6.9 8.4 3.8 Neo1 AD-48276 17.8 9.8 6.8 2.0 Neo1 AD-48269 18.4 5.510.9 4.4 Neo1 AD-48286 21.4 3.8 11.7 2.1 Neo1 AD-48299 22.9 3.0 11.7 3.8Neo1 AD-48295 36.2 16.3 12.0 0.4 Neo1 AD-48292 44.3 6.8 14.8 2.2 Neo1AD-48293 44.7 14.1 30.7 1.8 Neo1 AD-48288 46.9 21.9 31.9 5.2 Neo1AD-48307 50.2 10.1 16.8 3.9 Neo1 AD-48270 54.2 10.6 65.9 42.5 Neo1AD-48300 54.6 0.1 18.6 1.9 Neo1 AD-48306 56.6 19.5 16.0 2.3 Neo1AD-48315 57.7 3.5 17.6 8.0 Neo1 AD-48291 60.2 12.0 35.2 6.4 Neo1AD-48272 61.9 4.1 25.2 3.2 Neo1 AD-48271 62.6 4.7 35.4 6.8 Neo1 AD-4829462.6 2.1 22.7 11.0 Neo1 AD-48278 62.9 13.8 27.4 1.3 Neo1 AD-48277 63.220.4 26.1 2.6 Neo1 AD-48313 68.2 18.7 43.7 2.2 Neo1 AD-48289 70.6 15.353.6 12.3 Neo1 AD-48290 73.8 22.6 60.0 3.9 Neo1 AD-48284 74.0 19.2 106.943.7 Neo1 AD-48298 76.0 6.9 75.4 19.3 Neo1 AD-48311 77.9 22.6 23.5 11.1Neo1 AD-48285 81.0 11.5 65.3 14.2 Neo1 AD-48282 82.7 16.3 47.0 15.3 Neo1AD-48302 83.3 3.1 32.8 6.7 Neo1 AD-48303 85.0 16.3 29.2 7.7 Neo1AD-48279 90.2 6.2 51.7 14.3 Neo1 AD-48301 91.8 8.5 88.2 11.1 Neo1AD-48314 96.7 16.7 128.8 37.8 Neo1 AD-48312 107.9 30.0 94.0 27.8 Neo1AD-48304 111.6 22.3 91.6 33.2 Neo1 AD-48310 118.0 36.4 118.8 29.0

TABLE 21 BMP6 Duplexes Table 21 duplexName sOligoSeq SEQ ID NOasOligoSeq SEQ ID NO Set AD-47955.1 GcAGAAuuccGcAu 1242 UGuAGAUGCGGAAUU1300 humanRhesus cuAcAdTsdT CUGCdTsdT AD-47957.1 GAAuAuGGuuGuA 1243AGCUCUuAcAACcAuA 1301 humanRhesus AGAGcudTsdT UUCdTsdT AD-47966.1cucuucAuGcuGGA 1244 AcAGAUCcAGcAUGAA 1302 humanRhesus ucuGudTsdTGAGdTsdT AD-47989.1 GAGuucAAGuucA 1245 AuAAGUUGAACUUGA 1303 humanRhesusAcuuAudTsdT ACUCdTsdT AD-47993.1 cGuGAGuAGuuGu 1246 AGACcAAcAACuACUc1304 humanRhesus uGGucudTsdT ACGdTsdT AD-47960.1 GGAcGAccAuGAG 1247UuAUCUCUcAUGGUC 1305 humanRhesus AGAuAAdTsdT GUCCdTsdT AD-47997.1ccuAGAuuAcAucu 1248 AAGGcAGAUGuAAUC 1306 humanRhesus GccuudTsdTuAGGdTsdT AD-47985.1 cAAcAGAGucGuA 1249 AGCGAUuACGACUCU 1307 humanRhesusAucGcudTsdT GUUGdTsdT AD-47983.1 GucuAucAAAGGu 1250 AAAUCuACCUUUGAu 1308humanRhesus AGAuuudTsdT AGACdTsdT AD-47954.1 cccGGAcGAccAuG 1251UCUCUcAUGGUCGUC 1309 humanRhesus AGAGAdTsdT CGGGdTsdT AD-47972.1cucGucAGcGAcAc 1252 UUGUGGUGUCGCUG 1310 humanRhesus cAcAAdTsdTACGAGdTsdT AD-47981.1 ccAcuAAcucGAAA 1253 UCUGGUUUCGAGUuA 1311humanRhesus ccAGAdTsdT GUGGdTsdT AD-47982.1 GuAAAuGAcGuGA 1254AACuACUcACGUcAUU 1312 humanRhesus GuAGuudTsdT uACdTsdT AD-47987.1GGGGAcAcAcAuuc 1255 AGGcAGAAUGUGUGU 1313 humanRhesus uGccudTsdTCCCCdTsdT AD-47994.1 cGGcuGcAGAAuuc 1256 AUGCGGAAUUCUGcA 1314humanRhesus cGcAudTsdT GCCGdTsdT AD-47973.1 GccGAcAAcAGAG 1257UuACGACUCUGUUGU 1315 humanRhesus ucGuAAdTsdT CGGCdTsdT AD-47975.1GGAuGccAcuAAcu 1258 UUUCGAGUuAGUGGc 1316 humanRhesus cGAAAdTsdTAUCCdTsdT AD-47979.1 ccGAcAAcAGAGuc 1259 AUuACGACUCUGUUG 1317humanRhesus GuAAudTsdT UCGGdTsdT AD-47996.1 cGuGcuGuGcGccA 1260UuAGUUGGCGcAcAGc 1318 humanRhesus AcuAAdTsdT ACGdTsdT AD-47968.1cAAcGcAcAcAuGA 1261 UGcAUUcAUGUGUGC 1319 humanRhesus AuGcAdTsdTGUUGdTsdT AD-47977.1 cuGucuAucAAAGG 1262 AUCuACCUUUGAuAG 1320humanRhesus uAGAudTsdT AcAGdTsdT AD-47995.1 GcGGGucuccAGu 1263UGAAGcACUGGAGAC 1321 humanRhesus GcuucAdTsdT CCGCdTsdT AD-47959.1cuGAGuuuGGAuG 1264 uAcAGAcAUCcAAACUc 1322 humanRhesus ucuGuAdTsdTAGdTsdT AD-47962.1 cAGGAAGcAuGAG 1265 AuAcAGCUcAUGCUUC 1323 humanRhesuscuGuAudTsdT CUGdTsdT AD-47967.1 GGcuGGcuGGAAu 1266 UGUcAAAUUCcAGCcA 1324humanRhesus uuGAcAdTsdT GCCdTsdT AD-47986.1 GcAGAccuuGGuuc 1267AAGGUGAACcAAGGU 1325 humanRhesus AccuudTsdT CUGCdTsdT AD-47988.1GAcGuGAGuAGuu 1268 ACcAAcAACuACUcACG 1326 humanRhesus GuuGGudTsdTUCdTsdT AD-47990.1 cAGAGucGuAAuc 1269 uAGAGCGAUuACGAC 1327 humanRhesusGcucuAdTsdT UCUGdTsdT AD-47991.1 cAGAccuuGGuucA 1270 uAAGGUGAACcAAGG1328 humanRhesus ccuuAdTsdT UCUGdTsdT AD-47956.1 GGGucuccAGuGcu 1271UCUGAAGcACUGGAG 1329 humanRhesus ucAGAdTsdT ACCCdTsdT AD-47974.1GcAcAcAuGAAuGc 1272 UGGUUGcAUUcAUGU 1330 humanRhesus AAccAdTsdTGUGCdTsdT AD-47976.1 GGuAAAuGAcGuG 1273 ACuACUcACGUcAUUu 1331humanRhesus AGuAGudTsdT ACCdTsdT AD-47980.1 cAcAcAuGAAuGcA 1274UUGGUUGcAUUcAUG 1332 humanRhesus AccAAdTsdT UGUGdTsdT AD-47984.1cGAcAccAcAAAGA 1275 UGAACUCUUUGUGGU 1333 humanRhesus GuucAdTsdTGUCGdTsdT AD-47964.1 cucAuuAAuAAuuu 1276 UGAGcAAAUuAUuAA 1334humanRhesus GcucAdTsdT UGAGdTsdT AD-47970.1 cAuuAAuAAuuuGc 1277AGUGAGcAAAUuAUu 1335 humanRhesus ucAcudTsdT AAUGdTsdT AD-47971.1GuAcuGucuAucAA 1278 uACCUUUGAuAGAcAG 1336 humanRhesus AGGuAdTsdTuACdTsdT AD-47963.1 cuuGuGGAuGccAc 1279 AGUuAGUGGcAUCcAc 1337humanRhesus uAAcudTsdT AAGdTsdT AD-47965.1 GuucAGuAcuGucu 1280UUGAuAGAcAGuACU 1338 humanRhesus AucAAdTsdT GAACdTsdT AD-47992.1cuuGGAuuccuAGA 1281 UGuAAUCuAGGAAUCc 1339 humanRhesus uuAcAdTsdTAAGdTsdT AD-47998.1 GGucuGuAGcAAG 1282 ACUcAGCUUGCuAcAG 1340 humanRhesuscuGAGudTsdT ACCdTsdT AD-47958.1 GAuuuuAAAGGAc 1283 AAUGAGGUCCUUuAA 1341humanRhesus cucAuudTsdT AAUCdTsdT AD-47961.1 cAAAcuuuucuuAu 1284UGCUGAuAAGAAAAG 1342 humanRhesus cAGcAdTsdT UUUGdTsdT AD-47969.1GuGGAuGccAcuA 1285 UCGAGUuAGUGGcAU 1343 humanRhesus AcucGAdTsdTCcACdTsdT AD-47978.1 GucAGcGAcAccAc 1286 UCUUUGUGGUGUCGC 1344humanRhesus AAAGAdTsdT UGACdTsdT AD-47305.1 ucAuGAGcuuuGu 1287AGGUUcAcAAAGCUcA 1345 humanRhesus GAAccudTsdT UGAdTsdT Mouse AD-47325.1GAGAcGGcccuuAc 1288 UUGUCGuAAGGGCCG 1346 humanRhesus GAcAAdTsdTUCUCdTsdT Mouse AD-47329.1 AcGGcccuuAcGAc 1289 UGCUUGUCGuAAGGG 1347humanRhesus AAGcAdTsdT CCGUdTsdT Mouse AD-47309.1 AAccuGGuGGAGu 1290UGUCGuACUCcACcAG 1348 humanRhesus AcGAcAdTsdT GUUdTsdT Mouse AD-47317.1GcAGAGAcGGcccu 1291 UCGuAAGGGCCGUCU 1349 humanRhesus uAcGAdTsdTCUGCdTsdT Mouse AD-47313.1 AccuGGuGGAGuA 1292 UUGUCGuACUCcACcA 1350humanRhesus cGAcAAdTsdT GGUdTsdT Mouse AD-47321.1 AGAGAcGGcccuuA 1293UGUCGuAAGGGCCGU 1351 humanRhesus cGAcAdTsdT CUCUdTsdT Mouse AD-47333.1ucccAcucAAcGcAc 1294 AUGUGUGCGUUGAG 1352 humanRhesus AcAudTsdTUGGGAdTsdT Mouse AD-48038.1 ucAAcGAcGcGGAc 1295 ACcAUGUCCGCGUCG 1353mouseRat AuGGudTsdT UUGAdTsdT AD-48010.1 GccAucucGGuucu 1296AGuAAAGAACCGAGA 1354 mouseRat uuAcudTsdT UGGCdTsdT AD-48042.1AAuGccAucucGGu 1297 AAAGAACCGAGAUGG 1355 mouseRat ucuuudTsdT cAUUdTsdTAD-48000.1 AAcGAcGcGGAcA 1298 UGACcAUGUCCGCGU 1356 mouseRat uGGucAdTsdTCGUUdTsdT AD-48004.1 AuGccAucucGGuu 1299 uAAAGAACCGAGAUG 1357 mouseRatcuuuAdTsdT GcAUdTsdT It should be noted that unmodified versions of eachof the modified sequences shown are included within the scope of theinvention.

1. A double-stranded ribonucleic acid (dsRNA) for inhibiting expressionof hepcidin antimicrobial peptide (HAMP), wherein said dsRNA is selectedfrom the dsRNAs listed in Table 2, 3, 4, or 5 with a start position of382, 380, 379, or
 385. 2. The dsRNA of claim 1, wherein the dsRNAconsists of AD-48141, wherein the sense strand of AD-48141 isGAAcAuAGGucuuGGAAuAdTdT and the antisense strand of AD-48141 isUAuUCcAAGACCuAuGuUCdTdT.
 3. A dsRNA for inhibiting expression of HAMP,wherein said dsRNA comprises a sense strand and an antisense strand,wherein each strand is 30 nucleotides in length or less, and wherein theantisense strand comprises at least 15 contiguous nucleotides differingby no more than 3 nucleotides from one of the antisense strand sequenceslisted in Table 2, 3, 4, or
 5. 4.-13. (canceled)
 14. The dsRNA of claim3, wherein the sense strand comprises one of the sense strand sequencesof Table 2, 3, 4, or 5 and the antisense strand comprises one of theantisense strand sequences of Table 2, 3, 4, or
 5. 15. The dsRNA ofclaim 3, wherein the sense strand consists of one of the sense strandsequences of Table 2, 3, 4, or 5 and the antisense strand consists ofone of the antisense strand sequences of Table 2, 3, 4, or
 5. 16.(canceled)
 17. The dsRNA of claim 3, wherein said dsRNA furthercomprises at least one modified nucleotide.
 18. The dsRNA of claim 17,wherein at least one of said modified nucleotides is chosen from thegroup consisting of: a 2′-O-methyl modified nucleotide, a nucleotidecomprising a 5′-phosphorothioate group, and a terminal nucleotide linkedto a cholesteryl derivative or dodecanoic acid bisdecylamide group. 19.The dsRNA of claim 17, wherein said modified nucleotide is chosen fromthe group consisting of: a 2′-fluoro modified nucleotide, a 2′-fluoromodified nucleoside, a 2′-deoxy-2′-fluoro modified nucleotide, a2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide,2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholinonucleotide, a phosphoramidate, and a non-natural base comprisingnucleotide. 20.-23. (canceled)
 24. The dsRNA of claim 3, furthercomprising a ligand.
 25. The dsRNA of claim 24, wherein the ligand isconjugated to the 3′ end of the sense strand of the dsRNA.
 26. The dsRNAof claim 3, further comprising an N-Acetyl-Galactosamine (GalNac)conjugate.
 27. The dsRNA of claim 3, wherein the dsRNA is formulated ina nucleic acid lipid particle formulation.
 28. The dsRNA of claim 27,wherein the nucleic acid lipid particle formulation is selected fromTable A.
 29. The dsRNA of claim 27, wherein the nucleic acid lipidparticle formulation comprises MC3. 30.-32. (canceled)
 33. Apharmaceutical composition for inhibiting expression of a HAMP genecomprising the dsRNA of claim
 3. 34.-103. (canceled)
 104. A method ofinhibiting HAMP expression in a cell, the method comprising: (a)contacting the cell the dsRNA of claim 3; and (b) maintaining the cellproduced in step (a) for a time sufficient to obtain degradation of themRNA transcript of a HAMP gene, thereby inhibiting expression of theHAMP gene in the cell.
 105. (canceled)
 106. The method of claim 104,wherein the HAMP expression is inhibited by at least 30% or at least80%.
 107. A method of treating a disorder associated with HAMPexpression comprising administering to a subject in need of suchtreatment a therapeutically effective amount of the dsRNA of claim 3.108.-110. (canceled)
 111. The method of claim 107, wherein the subjecthas anemia, refractory anemia, anemia of chronic disease (ACD), oriron-restricted erythropoiesis.
 112. (canceled)
 113. The method of claim107, wherein the dsRNA is administered at a concentration of 0.01mg/kg-5 mg/kg bodyweight of the subject. 114.-272. (canceled)